Patent Grant
10758886
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 28, 2016, is named 42206-708_201_SL.txt and is 3,456 bytes in size.
Array technologies allow for large-scale, quantitative analyses of biological samples. However, the density and robustness of such technologies should be improved to allow increased sensitivity, reproducibility and accuracy of biological assays using such arrays.
Disclosed herein are methods and devices for making an array, the method comprising (a) performing a conditioning step on a surface of the array in the absence of monomer; (b) repeating step (a) at least once; (c) performing a synthesis step upon the surface to add at least one monomer; and (d) repeating step (c) at least once to form a sequence, wherein the conditioning step performed prior to synthesizing the sequence enhances attachment of the sequence to the surface of the array.
In some embodiments, the method comprises the conditioning step to be performed at least 5 times, at least 10 times or at least 15 times. In other embodiments, the binding of a target to a sequence is enhanced by the methods and devices disclosed herein by least 10%, at least 20%, at least 30%, at least 40% or at least 50%. In yet other embodiments, the binding of a target to a sequence is enhanced by the methods and devices disclosed herein by least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or at least 15-fold. In still other embodiments, the sensitivity of the array is enhanced by the methods and devices disclosed herein by at least 10%, at least 20%, at least 30%, at least 40% or at least 50%. In still other embodiments, the sensitivity of the array is enhanced by the methods and devices disclosed herein by at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or at least 15-fold.
Also disclosed herein are methods and devices for making an array, comprising (a) performing a chemical reaction upon the array that is capable of attaching a monomer to a coupling site, wherein the monomer forms part of a linker sequence; (b) performing an additional chemical reaction upon the array that is capable of adding a monomer to the attached monomer of step (a); (c) repeating step (b) at least once until the monomers that comprise the desired linker sequence have been added; and d. synthesizing in situ a functional sequence onto the free end of the linker sequence, wherein the linker sequence enhances binding between the functional sequence and a substrate.
In some embodiments, the methods and devices disclosed herein to make an array include a linker sequence between 2 and 5 monomers, between 6 and 10 monomers, between 11 and 15 monomers, between 16 and 20 monomers, between 21 and 25 monomers, between 26 and 30 monomers, or at least 30 monomers. In yet other embodiments, the linker sequences is at least 10% homogeneous, at least 20% homogeneous, at least 30% homogeneous, at least 40% homogeneous, at least 50% homogeneous, at least 60% homogeneous, at least 70% homogeneous, at least 80% homogeneous, at least 90% homogeneous, or at least 100% homogeneous.
In still other embodiments, the linker sequence has a net negative charge at a pH of 7. In other embodiments, the linker sequence has a net positive charge at a pH of 7. In yet other embodiments, the linker sequence has a neutral charge at a pH of 7.
In other embodiments, the linker sequence is composed of molecules selected from the group consisting of nucleotides, oligonucleotides, polynucleotides, ribonucleotides, oligoribonucleotides, polyribonucleotides, monosaccharides, oligosaccharides, polysaccharides, amino acids, peptides, polypeptides, proteins, lipids, synthetic or non-natural compounds, and peptide nucleic acids (PNAs). In additional embodiments, the linker sequence is composed of amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, selenocysteine, pyrrolysine, N-formylmethionine, L-theanine, gamma amino butyric acid (GABA), ornithine, citrulline, α-methylnorvaline, β-alanine, δ-aminolevulinic acid, and 4-aminobenzoic acid (PABA). In other embodiments, the linker sequence is homogeneously composed of glycine. In yet other embodiments, the linker sequence comprises at least one monomer unit of polyethylene glycol (PEG), diamines, diacids, amino acids, alcohols, thiols, or combinations thereof.
Also disclosed herein are arrays comprising: a. a surface, and b. a plurality of molecules, wherein the molecules comprise a linker sequence comprised of monomers and having two ends, wherein one end is coupled to the surface and the other end is coupled to a functional sequence which binds a target molecule. Also disclosed herein are arrays comprising: (a) a surface, and (b) a plurality of in situ synthesized molecules immobilized to the surface, wherein each molecule comprises a linker attached to a functional sequence, the linker being at least 1 monomer in length and interposed between the surface of the array and the functional sequence, and the functional sequence being at least 2 monomers in length.
Disclosed herein are peptide arrays comprising: (a) a surface, and (b) a plurality of peptides immobilize to the surface, wherein each peptide comprises a linker sequence attached to a functional sequence, the linker sequence being interposed between the surface of the array and the functional sequence. Also disclosed herein are methods of making an array of molecules, comprising: a. performing a first chemical reaction on the array, wherein a pre-synthesized linker is attached to a surface of the array; and b. performing a second chemical reaction upon the array, wherein a pre-synthesized functional sequence is attached to the linker sequence, wherein the linker sequence enhances binding between the functional sequence and a target.
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
Arrays can be generated using methods that include immobilizing purified molecules or monomers onto suitable surfaces or synthesizing them in situ directly on the surface. In situ synthesis offers significant advantages over other methods such as a greater number and density of features, and enhanced sensitivity, but the quality and reliability of in situ arrays can be contingent on the quality and consistency of the synthesized molecules. One important aspect of an in situ synthetic process is the preparation of the surface upon which in situ synthesis is performed. Factors that are important to in situ array synthesis can include the proper preparation of the surface, the availability of attachment groups that can be used as a base for attachment, the density of those groups, the tendency of the surface or matrix surrounding the attachment groups to interfere with the function of the final synthesized molecule, for example through nonspecific binding or by modifying the synthesized molecule to interact with a molecular partner of choice, and the evenness of the distribution of attachment groups of multiple length.
Different methods exist for placing attachment groups on surfaces. One of the most common surface modifications, particularly for surfaces that have a large fraction of silicon oxide, is to use a functionalized silane, for example an amino silane. Depending on the chemistry of attachment, such molecules can add to the surface in a well-defined layer or as a more complex cross-linked structure. In situ synthesis steps can then be performed upon the attachment groups on the surface by exposing them to monomers in reactive coupling steps that link them together.
The present disclosure recognizes the advantages of preparing conditioned surfaces for in situ molecular array synthesis. Some arrays, such as the arrays described in PCT/US2013/065541 and U.S. Pat. No. 5,424,186, hereinafter incorporated by reference in their entireties, provide a method of analyzing various materials including biological samples. However, the sensitivity and consistency of array-based detection can depend upon the properties of the attachment groups and the surface on which they reside.
Recognized herein is that molecules of previously described arrays are not all equally available for binding on the array. Such variability can lead to differences in sensitivity between features on or between arrays, and thus reproducibility of biological assay results on these arrays. The present disclosure recognizes that compensation can be made for this variability by increasing or growing a linker molecule or molecules on the attachment sites. Accordingly, included herein are methods to modify or increase the length of linkers on attachment sites of the arrays in order to optimize binding of targets in the biological samples to molecules attached to the array.
Also recognized herein is that the attachment groups of previously described arrays are not all equally available for reactive coupling. Moreover, the present disclosure recognizes that the surface material of an array is not entirely functionally inert (both in terms of the synthesis and the final function of the molecule) at locations other than the chemical attachment site. The present disclosure also recognizes that the structure of the silane or other attachment matrices can be affected by the synthetic process. As a consequence, some in situ synthesis methods may lead to the generation of surfaces with different properties in earlier synthetic steps versus later synthetic steps.
Described herein are methods for in situ synthesis of arrays that significantly enhances attachment of a sequence to the surface of the array. These methods allow for the production of arrays with enhanced sensitivity that are applicable to a wide variety of applications, including but not limited to: diagnostic arrays, arrays for selection of specific ligands or targets, arrays for potential drugs, and arrays of sensor molecules. In some cases, the approach can comprise performing multiple cycles (or partial cycles) of surface conditioning prior to the main synthesis, thereby allowing a surface to become stabilized before the molecules or sequences are actually in situ synthesized, or otherwise incorporated, into the array. The result can be an array wherein the molecules or sequences bind to their ligands or targets with greater sensitivity and reduced background noise.
In some cases, the cycles are run in such a way that no coupling of a new molecular component to the system can occur prior to the main synthesis. In other cases, the cycles are run in such a way that coupling of a new molecular component to the system can occur prior to the main synthesis. In yet other cases, the process involves a combination of cycles that couple new molecular components to the system and cycles that do not couple new molecular components to the system prior to the main synthesis. In some instances, the process involves growing a “linker” on the attachment sites that is attached to a molecule or sequence prior to the main synthesis.
Also provided herein is method of providing a conditioned surface to an array. In some cases, the surface comprises a number of in situ synthesized molecules attached to a conditioned surface. The surface can comprise a number of molecules that have been synthesized in situ or otherwise added to the array.
Terms that are not defined in the present application or any incorporated references will be given their plain and ordinary meaning in the field as understood by one of ordinary skill in the art.
As used in the present specification, the following words, phrases and symbols are generally intended to have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise. The following abbreviations and terms have the indicated meanings throughout:
As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an array” may include a plurality of arrays unless the context clearly dictates otherwise.
An “array” refers to an intentionally created collection of molecules or sequences attached to or fabricated on a substrate or surface in which the identity or source of a group of molecules is known based on its location on the array. The molecules or sequences housed on the array and within a feature of an array can be identical to or different from each other.
Molecules can include nucleotides, oligonucleotides, polynucleotides, ribonucleotides, oligoribonucleotides, polyribonucleotides, monosaccharides, oligosaccharides, polysaccharides, amino acids, peptides, polypeptides, proteins, lipids, synthetic or non-natural compounds, peptide nucleic acids (PNAs) and other materials known in the art. In some embodiments, the molecules are a plurality of monomers that form a polymeric sequence. In other embodiments, the polymeric sequences bind to or recognize a target, e.g., ligand or binding partner, in a sample or specimen. In yet other embodiments, the molecules or polymeric sequences may include a linker as described herein.
Monomers can include nucleotides, monosaccharides, amino acids, and other subunit components. Monomer molecules can include natural amino acids, non-natural amino acids, nucleic acids, and analogues thereof.
A polymer can comprise two or more linked monomers. The monomers in a particular polymeric linker are not necessarily identical and can be of heterogeneous composition, comprising different monomers of the same class of molecules.
A class of molecules can be each of the following: nucleotides, oligonucleotides, polynucleotides, ribonucleotides, oligoribonucleotides, polyribonucleotides, monosaccharides, oligosaccharides, polysaccharides, amino acids, peptides, polypeptides, proteins, lipids, synthetic or non-natural compounds, or peptide nucleic acids (PNAs).
Polymers can be copolymers such as alternating copolymers, periodic copolymers, statistical copolymers, or block copolymers. Polymers can comprise linear or branched structures. Branched structures can result when one or more substituents or side groups on a parent polymeric chain are replaced by another chain of a polymer. The substituted polymeric chain or branch or side chain can be comprised of the same class or classes of monomers, or of different class or classes of monomers from the parent polymeric chain. In some branched structures, the side chains can grow off the parent chain at regular intervals such as every 10 monomeric subunits. In others, the side chains can grow off the parent chain at irregular intervals. A branched polymer can be a graft polymer, a star-shaped polymer, a comb polymer, or a dendrimer. Polymers can also form a polymer network in which all polymer chains are interconnected through branching and/or crosslinking to form a macroscopic entity. Crosslinking can occur between branches on the same polymer or between branches of distinct polymers. Where linear and branched polymers are in proximity, crosslinking can also occur between those linear and branched polymers. The three-dimensional structure of a polymer can be lacking a defined structure. For example, the polymer may be a flexible loop that lacks active sites capable of bonding or interacting with other sites on that same loop or with other molecules, which can cause its orientation and shape to continually change. Conversely, a polymer can form a more defined structure. For example, a peptide polymer may interact with itself or with neighboring polymers to form secondary, tertiary, and/or quaternary structures.
Main synthesis can comprise the synthesis steps that couples monomers to an attachment group on the surface or to other molecules already coupled to the attachment group. The added monomer can comprise a functional sequence or an inert sequence.
A “linker” can be either sequentially or non-sequentially incorporated into an array. A linker can be added to a growing polymer, or vice versa. A linker can be a component that elongates the distance between the substrate surface of an array and a molecule, such as a polypeptide or polynucleotide. A “linker sequence” can be a polymer of linker components. A “linker” can mean a linker component (including but not limited to a nucleotide, an amino acid or a chemical constituent), a linker sequence, and/or a linker polymer.
A “functional sequence” or “functional molecule” can recognize or bind to a target, e.g, ligand or binding partner to the functional sequence or functional molecule.
A “target” is a ligand or binding partner which can bind or interact with one or more functional molecules or sequences on the surface of an in situ synthesized array.
Couple or coupling comprises forming a chemical bond between two components, including but not limited to two molecules or sequences, or to a molecule or sequence and a linker, or to a molecule and an attachment site. The coupling moieties includes but is not limited to phosphodiester or amide bonds, ester bonds, thioester bonds, ether bonds, and carbon-carbon bonds.
Attach or attachment is equivalent to couple or coupling. An attachment site is a chemical or component that is capable of forming a chemical bond with another molecule. This includes but is not limited to phosphodiester or amide bonds, ester bonds, thioester bonds, ether bonds, and carbon-carbon bonds.
A surface can be a “solid support,” “support,” and “substrate,” that serves as a physical support for a polymer or a group of polymers.
A molecule can be attached to a surface directly or via a linker as disclosed herein. Direct attachment is possible by covalent attachment of a molecule, such as an amino acid to a region or feature of the surface. For example, the linker(s) as disclosed herein could be attached to a chemically reactive or chemically inert region of the surface. Optionally or additionally, the linkers may be linear or branched, having at least one or a multiplicity of reactive sites for attachment of a molecule as described herein. A linker can be cleavable, non-cleavable, self-immolating, hydrophilic, or hydrophobic. A linker as disclosed herein can also be flexible or rigid, or a combination thereof, which may be dependent upon the specific linker sequence employed. For example, glycine-rich linkers tend to be flexible, whereas proline-rich linkers tend to form relatively rigid extended structures.
A linker as disclosed herein can be a component that is sequentially or non-sequentially incorporated into an array. For example, a linker as disclosed herein may be formed on the surface of an array by sequentially attaching a linker component to an attachment site (e.g., aminosilane) followed by additional linker components that are added to the growing polymer. Alternatively, a linker polymer as disclosed herein may be prefabricated or pre-synthesized and then attached to the array surface without in situ sequential addition of linker molecules. Likewise, as disclosed herein a pre-synthesized molecule or sequence may be attached to an in situ synthesized linker or a pre-synthesized linker. In addition, as disclosed herein a combination of prefabricated linker polymers and in situ synthesis of additional linkers may be utilized to generate the finished linker polymer on the array surface.
In some embodiments, a linker as disclosed herein can be divided into separate linkers or sub-linkers by an intervening functional molecule or functional sequence. For example, a first linker or sub-linker may be attached to the attachment group on the array surface on one end, wherein an intervening functional sequence is joined or attached to the free end of the first linker, and a second linker may be then joined or attached to the free end of the intervening functional sequence. A second intervening functional sequence may then be joined to the free end of the second linker, and so on and so forth, wherein a polymer is generated that comprises a chain of alternating linker sequences and functional sequences.
A linker as disclosed herein can comprise a single monomer linked together, or it can comprise two or more linked monomers. Thus, the monomers in a particular polymeric linker are not necessarily identical and can be of heterogeneous composition, comprising different monomers of the same class. For example, valine and glycine are different monomers within the same class of amino acids. Alternatively, linkers may comprise monomers that belong to different classes. For example, a linker as disclosed herein may comprise amino acids from different classes (e.g., acidic and basic amino acids) or amino acids and nucleotides. Additionally, a linker as disclosed herein may also comprise modified amino acids, including but not limited to amino acids modified with polyethylene glycol, long-chain amine, long-chain alcohol or polyene derivative.
Linkers as disclosed herein can be copolymers such as alternating copolymers, periodic copolymers, statistical copolymers, or block copolymers. Linkers as disclosed herein can comprise linear or branched structures. Branched structures can result when one or more substituents or side groups on a parent polymeric chain are replaced by another chain of a polymer. The substituted polymeric chain or branch or side chain can be comprised of the same class or classes of monomers, or of different class or classes of monomers from the parent polymeric chain that forms part of the linker. In some branched structures, the side chains can grow off the parent chain at regular intervals such as every 10 monomeric subunits. In others, the side chains can grow off the parent chain at irregular intervals. A branched polymer linker as disclosed herein can be a graft polymer, a star-shaped polymer, a comb polymer, or a dendrimer. Linkers as disclosed herein can also form a polymer network in which all polymer chains are interconnected through branching and/or crosslinking to form a macroscopic entity. Crosslinking can occur between branches on the same linker or between branches of distinct linkers. Where linear and branched linkers are in proximity, crosslinking can also occur between those linear and branched linkers. The three-dimensional structure of a linker as disclosed herein can be lacking a defined structure. For example, the linker as disclosed herein may be a flexible loop that lacks active sites capable of bonding or interacting with other sites on that same loop or with other linker components, which can cause its orientation and shape to continually change. Conversely, a linker as disclosed herein can form a more defined structure. For example, a peptide linker as disclosed herein may interact with itself or with neighboring components to form secondary, tertiary, and/or quaternary structures.
The linkers as disclosed herein may be, for example, aryl acetylene, ethylene glycol oligomers containing 2-20 monomer units, 2-10 monomer units or 1-5 monomer units including but not limited to polyethylene glycol (PEG), diamines, diacids, amino acids, alcohols, thiols, and combinations thereof. Examples of diamines include ethylene diamine and diamino propane. Alternatively, the linkers as disclosed herein may be the same type as that being synthesized (i.e., nascent polymers), such as polypeptides and polymers of amino acid derivatives such as for example, amino hexanoic acids. While most natural amino acids are alpha amino acids, linkers can comprise monomeric subunits selected from alpha, beta, gamma, delta, and/or epsilon amino acids. Linkers as disclosed herein may comprise amino acid monomers in either the L or D isomeric forms. Linkers as disclosed herein can include monomeric subunits selected from the 20 standard proteinogenic amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, the amino acids C, I, T and M, and optionally Q and E, are not included as subunits in the linker. Linkers as disclosed herein may also have monomeric subunits selected from non-standard proteinogenic amino acids such as selenocysteine, pyrrolysine, and N-formylmethionine. Linkers may also have monomeric subunits selected from non-proteinogenic amino acids, which can include L-theanine, gamma amino butyric acid (GABA), ornithine, citrulline, α-methylnorvaline, β-alanine, δ-Aminolevulinic acid, and/or 4-Aminobenzoic acid (PABA). Similarly, the linkers may comprise polymers as defined in this specification. For example, linkers as disclosed herein can comprise nucleic acids (DNA or RNA), oligosaccharides or polysaccharides, lipids, fatty acids, peptide nucleic acids, synthetic compounds, or analogues thereof.
Linkers as disclosed herein may be of homogeneous or heterogeneous composition. For example, in some embodiments a linker as disclosed herein may be a peptide linker homogeneously composed of only alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, selenocysteine, pyrrolysine, N-formylmethionine, L-theanine, gamma amino butyric acid (GABA), ornithine, citrulline, α-methylnorvaline, β-alanine, β-Aminolevulinic acid, or 4-Aminobenzoic acid (PABA) monomers. In other embodiments, a peptide linker as disclosed herein may be heterogeneously composed of more than one type of monomer selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, selenocysteine, pyrrolysine, N-formylmethionine, L-theanine, gamma amino butyric acid (GABA), ornithine, citrulline, α-methylnorvaline, β-alanine, δ-Aminolevulinic acid, and 4-Aminobenzoic acid (PABA) monomers. A heterogeneous linker as disclosed herein composition may be at least of a certain homogeneous percentage. For example, a linker composed of 50% Alanine, 10% Glycine, 10% Isoleucine, 10% Tryptophan, 10% Serine, and 10% Glutamine is 50% homogeneous. The highest percentage component is the homogeneous percentage.
In some embodiments, a linker as disclosed herein comprises a composition that is at least 10% homogeneous, 20% homogeneous, 30% homogeneous, 40% homogeneous, 50% homogeneous, 60% homogeneous, 70% homogeneous, 80% homogeneous, 90% homogeneous, or 100% homogeneous.
In some embodiments, a linker as disclosed herein comprises a composition that is no more than 10% homogeneous, 20% homogeneous, 30% homogeneous, 40% homogeneous, 50% homogeneous, 60% homogeneous, 70% homogeneous, 80% homogeneous, 90% homogeneous, or 100% homogeneous.
In some embodiments, a linker as disclosed herein comprises a composition that is 10% homogeneous, 20% homogeneous, 30% homogeneous, 40% homogeneous, 50% homogeneous, 60% homogeneous, 70% homogeneous, 80% homogeneous, 90% homogeneous, or 100% homogeneous.
The attachment(s) as disclosed herein between linkers or between a linker and an attachment site on an array surface can be via an amide bond, an ester bond, an ether bond, a carbamate bond, a carbon-nitrogen bond, a triazole, a macrocycle, an oxime bond, a hydrazone bond, immine bonds, a carbon-carbon single double or triple bond, a disulfide bond, thioester bond, or a thioether bond. Non-limiting examples of the functional groups for attachment include functional groups capable of forming, for example, an amide bond, an ester bond, an ether bond, a carbonate bond, a carbamate bond, or a thioether bond.
Non-limiting examples of functional groups capable of forming such bonds include amino groups; carboxylic acid groups, hydroxyl groups, carboxyl groups; aldehyde groups; azide groups; aniline groups, pyrrole groups, nitrile groups, isonitrile groups, aziridine groups, alkyne and alkene groups; ketones; hydrazides; acid halides such as acid fluorides, chlorides, bromides, and iodides; acid anhydrides, including symmetrical, mixed, and cyclic anhydrides; carbonates; carbonyl functionalities bonded to leaving groups such as cyano, succinimidyl, and N-hydroxysuccinimidyl; hydroxyl groups; sulfhydryl groups; and compounds and components possessing, for example, alkyl, alkenyl, alkynyl, allylic, or benzylic leaving groups, such as halides, mesylates, tosylates, triflates, epoxides, phosphate esters, sulfate esters, and besylates.
A linker as disclosed herein may have one or more functional groups, for example, one functional group that is bonded to the surface, and one functional group that is bonded to the growing chain (e.g.: peptide chain), and a linking portion between the two functional groups. Non-limiting examples of the linking portion include alkylene, alkenylene, alkynylene, polyenes, sugars, sugar polymers, nucleic acids, nucleic acid polymers, fatty acids, lipids, polyether, such as polyethylene glycol (PEG), polyester, polyamide, polyamino acids, polypeptides, cleavable peptides, valine-citrulline, substituted phenyl derivatives (for example, hydroxy benzoic acid), aminobenzylcarbamates, D-amino acids, and polyamine, any of which being unsubstituted or substituted with any number of substituents, such as halogens, hydroxyl groups, sulfhydryl groups, amino groups, nitro groups, nitroso groups, cyano groups, azido groups, sulfoxide groups, sulfone groups, sulfonamide groups, carboxyl groups, carboxaldehyde groups, imine groups, alkyl groups, halo-alkyl groups, alkenyl groups, halo-alkenyl groups, alkynyl groups, halo-alkynyl groups, alkoxy groups, aryl groups, aryloxy groups, aralkyl groups, arylalkoxy groups, heterocyclyl groups, acyl groups, acyloxy groups, carbamate groups, amide groups, urethane groups, epoxides, and ester groups.
Examples of peptide linkers as disclosed herein include the following sequences: GSG, GGSG (SEQ ID NO: 2), GGGSG (SEQ ID NO: 3), GGGGSG (SEQ ID NO: 4), GGGGGSG (SEQ ID NO: 5), and GGGGGGSG (SEQ ID NO: 6) (see, e.g., experiments shown on
In some embodiments, a linker as disclosed herein can maintain a net positive charge at a pH of about 0, a pH of about 1, a pH of about 2, a pH of about 3, a pH of about 4, a pH of about 5, a pH of about 6, a pH of about 7, a pH of about 8, a pH of about 9, a pH of about 10, a pH of about 11, a pH of about 12, a pH of about 13, or a pH of about 14.
In some embodiments, a linker as disclosed herein can maintain a net negative charge at a pH of about 0, a pH of about 1, a pH of about 2, a pH of about 3, a pH of about 4, a pH of about 5, a pH of about 6, a pH of about 7, a pH of about 8, a pH of about 9, a pH of about 10, a pH of about 11, a pH of about 12, a pH of about 13, or a pH of about 14.
In some embodiments, a linker as disclosed herein can maintain a net neutral charge at a pH of about 0, a pH of about 1, a pH of about 2, a pH of about 3, a pH of about 4, a pH of about 5, a pH of about 6, a pH of about 7, a pH of about 8, a pH of about 9, a pH of about 10, a pH of about 11, a pH of about 12, a pH of about 13, or a pH of about 14.
In some embodiments, a linker as disclosed herein can maintain a net positive charge at a pH above 0 and up to and including 1, a pH above 1 and up to and including 2, a pH above 2 and up to and including 3, a pH above 3 and up to and including 4, a pH above 4 and up to and including 5, a pH above 5 and up to and including 6, a pH above 6 and up to and including 7, a pH above 7 and up to and including 8, a pH above 8 and up to and including 9, a pH above 9 and up to and including 10, a pH above 10 and up to and including 11, a pH above 11 and up to and including 12, a pH above 12 and up to and including 13, or a pH above 13 and up to and including 14. In some embodiments, a linker can maintain a net negative charge at a pH above 0 and up to and including 1, a pH above 1 and up to and including 2, a pH above 2 and up to and including 3, a pH above 3 and up to and including 4, a pH above 4 and up to and including 5, a pH above 5 and up to and including 6, a pH above 6 and up to and including 7, a pH above 7 and up to and including 8, a pH above 8 and up to and including 9, a pH above 9 and up to and including 10, a pH above 10 and up to and including 11, a pH above 11 and up to and including 12, a pH above 12 and up to and including 13, or a pH above 13 and up to and including 14.
In some embodiments, a linker as disclosed herein can maintain a net neutral charge at a pH above 0 and up to and including 1, a pH above 1 and up to and including 2, a pH above 2 and up to and including 3, a pH above 3 and up to and including 4, a pH above 4 and up to and including 5, a pH above 5 and up to and including 6, a pH above 6 and up to and including 7, a pH above 7 and up to and including 8, a pH above 8 and up to and including 9, a pH above 9 and up to and including 10, a pH above 10 and up to and including 11, a pH above 11 and up to and including 12, a pH above 12 and up to and including 13, or a pH above 13 and up to and including 14.
In some embodiments, a linker as disclosed herein can reduce the background signal on an array by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%.
In some embodiments, a linker as disclosed herein can increase the sensitivity of the array by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.
In some embodiments, a linker as disclosed herein increases the sensitivity of the array by at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or at least 15-fold.
In some embodiments, a linker as disclosed herein increases the specificity of the array, as compared to an array with a decreased length or shorter linker, by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.
In some embodiments, a linker as disclosed herein increases the specificity of the array, as compared to an array with a decreased length or shorter linker, by at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or at least 15-fold.
In some embodiments, a linker as disclosed herein increases the reproducibility of the array, as compared to an array with a decreased length or shorter linker, by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.
In some embodiments, a linker as disclosed herein increases the reproducibility of the array by at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or at least 15-fold.
In some embodiments, the linker as disclosed herein increases the signal to background ratio of an array by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.
In some embodiments, the linker as disclosed herein increases the signal to background ratio of an array by at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or at least 15-fold.
In some embodiments, a linker as disclosed herein has a length of 2 monomers, 3 monomers, 4 monomers, 5 monomers, 6 monomers, 7 monomers, 8 monomers, 9 monomers, 10 monomers, 11 monomers, 12 monomers, 13 monomers, 14 monomers, 15 monomers, 16 monomers, 17 monomers, 18 monomers, 19 monomers, 20 monomers, 21 monomers, 22 monomers, 23 monomers, 24 monomers, 25 monomers, 26 monomers, 27 monomers, 28 monomers, 29 monomers, 30 monomers, 31 monomers, 32 monomers, 33 monomers, 34 monomers, 35 monomers, 36 monomers, 37 monomers, 38 monomers, 39 monomers, 40 monomers, 41 monomers 42 monomers, 43 monomers, 44 monomers, 45 monomers, 46 monomers, 47 monomers, 48 monomers, 49 monomers, or 50 monomers.
In some embodiments, a linker as disclosed herein has a length between 2 and 5 monomers, between 6 and 10 monomers, between 11 and 15 monomers, between 16 and 20 monomers, between 21 and 25 monomers, between 26 and 30 monomers, between 31 and 35 monomers, between 36 and 40 monomers, between 41 and 45 monomers, between 46 and 50 monomers, between 51 and 55 monomers, between 56 and 60 monomers, between 61 and 65 monomers, between 66 and 70 monomers, between 71 and 75 monomers, between 76 and 80 monomers, between 81 and 85 monomers, between 86 and 90 monomers, between 91 and 95 monomers, or between 95 and 100 monomers.
In some embodiments, a linker as disclosed herein has a length of at least 2 monomers, 3 monomers, 4 monomers, 5 monomers, 6 monomers, 7 monomers, 8 monomers, 9 monomers, 10 monomers, 11 monomers, 12 monomers, 13 monomers, 14 monomers, 15 monomers, 16 monomers, 17 monomers, 18 monomers, 19 monomers, 20 monomers, 21 monomers, 22 monomers, 23 monomers, 24 monomers, 25 monomers, 26 monomers, 27 monomers, 28 monomers, 29 monomers, 30 monomers, 31 monomers, 32 monomers, 33 monomers, 34 monomers, 35 monomers, 36 monomers, 37 monomers, 38 monomers, 39 monomers, 40 monomers, 41 monomers 42 monomers, 43 monomers, 44 monomers, 45 monomers, 46 monomers, 47 monomers, 48 monomers, 49 monomers, or 50 monomers.
In some embodiments, a particular linker sequence as disclosed herein is between 1 and 3 monomers in length, or between 4 and 6 monomers in length, or between 7 and 9 monomers in length, or between 10 and 12 monomers in length, or between 13 and 15 monomers in length, or between 16 and 18 monomers in length, or between 19 and 21 monomers in length, or between 22 and 24 monomers in length, or between 25 and 27 monomers in length, or between 28 and 30 monomers in length, or between 31 and 33 monomers in length, or between 34 and 36 monomers in length, or between 37 and 39 monomers in length, or between 40 and 42 monomers in length, or between 43 and 45 monomers in length, or between 46 and 48 monomers in length, or between 49 and 51 monomers in length, or between 52 and 54 monomers in length, or between 55 and 57 monomers in length, or between 58 and 60 monomers in length, or between 61 and 63 monomers in length, or between 64 and 66 monomers in length, or between 67 and 69 monomers in length, or between 70 and 72 monomers in length, or between 73 and 75 monomers in length, or between 76 and 78 monomers in length, or between 79 and 81 monomers in length, or between 82 and 84 monomers in length, or between 85 and 87 monomers in length, or between 88 and 90 monomers in length, or between 91 and 93 monomers in length, or between 94 and 96 monomers in length, or between 97 and 99 monomers in length, or between 100 and 102 monomers in length.
In some embodiments, a particular functional sequence as disclosed herein has a length of 1 monomer, 2 monomers, 3 monomers, 4 monomers, 5 monomers, 6 monomers, 7 monomers, 8 monomers, 9 monomers, 10 monomers, 11 monomers, 12 monomers, 13 monomers, 14 monomers, 15 monomers, 16 monomers, 17 monomers, 18 monomers, 19 monomers, or 20 monomers.
In some embodiments, a particular functional sequence as disclosed herein has a length of at least 1 monomer, 2 monomers, 3 monomers, 4 monomers, 5 monomers, 6 monomers, 7 monomers, 8 monomers, 9 monomers, 10 monomers, 11 monomers, 12 monomers, 13 monomers, 14 monomers, 15 monomers, 16 monomers, 17 monomers, 18 monomers, 19 monomers, or 20 monomers.
In some embodiments, a particular functional sequence as disclosed herein is between 1 and 3 monomers in length, or between 4 and 6 monomers in length, or between 7 and 9 monomers in length, or between 10 and 12 monomers in length, or between 13 and 15 monomers in length, or between 16 and 18 monomers in length, or between 19 and 21 monomers in length, or between 22 and 24 monomers in length, or between 25 and 27 monomers in length, or between 28 and 30 monomers in length, or between 31 and 33 monomers in length, or between 34 and 36 monomers in length, or between 37 and 39 monomers in length, or between 40 and 42 monomers in length, or between 43 and 45 monomers in length, or between 46 and 48 monomers in length.
In some embodiments, a combined linker and sequence as disclosed herein has a length of at least 4 monomers, 5 monomers, 6 monomers, 7 monomers, 8 monomers, 9 monomers, 10 monomers, 11 monomers, 12 monomers, 13 monomers, 14 monomers, 15 monomers, 16 monomers, 17 monomers, 18 monomers, 19 monomers, or 20 monomers.
In some embodiments, a combined linker and functional sequence as disclosed herein is between 1 and 3 monomers in length, or between 4 and 6 monomers in length, or between 7 and 9 monomers in length, or between 10 and 12 monomers in length, or between 13 and 15 monomers in length, or between 16 and 18 monomers in length, or between 19 and 21 monomers in length, or between 22 and 24 monomers in length, or between 25 and 27 monomers in length, or between 28 and 30 monomers in length, or between 31 and 33 monomers in length, or between 34 and 36 monomers in length, or between 37 and 39 monomers in length, or between 40 and 42 monomers in length, or between 43 and 45 monomers in length, or between 46 and 48 monomers in length.
In some embodiments, the linker sequences as disclosed herein present on a surface are of a uniform length. For example, in certain embodiments, photolithography masks may not be utilized, wherein linker synthesis is conducted upon the entire array surface to produce linkers of uniform length. In other embodiments, the linker sequences present on a surface as disclosed herein are not of uniform length. For example, non-uniform linker sequences on a surface may arise from the use of photolithography masks during synthesis that result in differential addition of monomeric subunits to distinct features on the array surface. In certain embodiments, the linkers within a given feature can be of uniform length, while having a distinct length from linkers in a different feature.
In some embodiments, the linker sequences as disclosed herein will be of one uniform length for a subset of features and of a different uniform length for at least one other subset of features on the given surface.
A surface as disclosed herein can refer to a material or group of materials having rigid or semi-rigid properties. A surface can comprise one or more materials having porous, permeable, or semi-permeable properties. A surface can also comprise one or more materials having malleable, ductile, brittle, plastic or elastic properties. A surface can also comprise one or more materials having polar, charged, acidic, or basic properties.
A surface as disclosed herein can be a flat surface, or a round or curved surface (such as a bead). In certain embodiments of a flat surface, the shape of that surface may be a circle, an oval, an ellipse, a crescent, a triangle, a curvlinear triangle, a quatrefoil, a parallelogram, a square, a rhombus, a trapezoid, a kite, a pentagon, a hexagon, a heptagon, an octagon, a nonagon, or a decagon. In certain embodiments of a round or curved surface, the surface may take on the shape of a sphere, an ellipsoid, or a more complex shape. In some embodiments, at least one surface of the solid support can be substantially flat.
A surface as disclosed herein can be homogenous or heterogeneous. A surface can comprise physical elements such as wells, raised regions, bumps, indentations, towers, pins, etched trenches, or combinations thereof. In certain embodiments, the physical elements on the surface can be of roughly uniform dimensions. In certain embodiments, the physical elements on the surface can be of non-uniform dimensions. In certain embodiments, the physical elements occur on the surface at discrete locations in a non-repetitive pattern. In certain embodiments, the physical elements occur on the surface in a repetitive pattern. In such embodiments, repetitive physical elements that are adjacent to one another can be separated by a uniform distance. Alternatively, the repetitive physical elements that are adjacent to one another can be separated by non-uniform distances.
In some embodiments, the solid support may be porous.
Non-limiting examples of substrate or surface materials as disclosed herein include, for example, silicon, biocompatible polymers such as, for example poly(methyl methacrylate) (PMMA) and polydimethylsiloxane (PDMS), glass, SiO, (such as, for example, a thermal oxide silicon wafer such as that used by the semiconductor industry), quartz, silicon nitride, functionalized glass, gold, platinum, metalloporphyrins, tungsten, silica, diamond, titanium, polyester, polyamide, polyimide, polyether, polysulfone, fluoropolymer, and aluminum.
In certain embodiments, as disclosed herein an organosilane is used to provide the surface attachment group. In certain embodiments, the organosilane comprises aminosilane, epoxysilane, or fluorosilane.
Non-limiting examples of functionalized surfaces as disclosed herein include: amino-functionalized glass, carboxy functionalized glass, hydroxy functionalized glass, aldehyde functionalized glass, Streptavidin functionalized glass, Neutravidin functionalized glass, Avidin functionalized glass, Poly-L-Lysine functionalized glass, pyridine functionalized glass, and 3-aminopropyl-triethoxysilane (APTS) functionalized glass.
Additionally, a surface may optionally be coated with one or more layers to provide a surface for molecular attachment or functionalization, increased or decreased reactivity, binding detection, or other specialized application. In such embodiments, as disclosed herein the surface may be optionally coated with at least 1 layer, at least 2 layers, at least 3 layers, at least 4 layers, at least 5 layers, at least 6 layers, at least 7 layers, at least 8 layers, at least 9 layers, at least 10 layers, at least 11 layers, at least 12 layers, at least 13 layers, at least 14 layers, at least 15 layers, at least 16 layers, at least 17 layers, at least 18 layers, at least 19 layers, or at least 20 layers. In certain embodiments, the layers coated on the surface can provide cumulative properties such as increased molecular attachment or functionalization, increased or decreased reactivity, binding detection, or other specialized application. In certain embodiments, the layers coated on the surface can provide discrete properties such as one layer providing molecular attachment for one category of monomers or polymers while another layer provides molecular attachment for a different category of monomers or polymers. For example, one layer may provide molecular attachment for peptides, while a second layer provides molecular attachment for nucleic acids to form a combination array.
Substrate or surface materials and or layer(s) may be porous or non-porous. For example, a substrate may be comprised of porous silicon. Furthermore, the substrate or surface materials may be permeable or semipermeable. Additionally, the substrate as disclosed herein may be a silicon wafer or chip such as those used in the semiconductor device fabrication industry. In the case of a wafer or chip, a plurality of arrays may be synthesized on the wafer. In some embodiments, the substrate is chosen from glass, silicon and silicon having a silicon oxide layer.
In some embodiments as disclosed herein, a method for in situ synthesis of the array is photolithography-based. In some embodiments, the photolithography-based synthesis comprises a photomask patterned step.
In some embodiments as disclosed herein, a method for the photolithography-based synthesis of the arrays uses a minimum number of photomasks to construct the array. In some embodiments, the array comprises features of about 0.5 micron to about 200 microns in diameter and a center-to-center distance of about 1 micron to about 300 microns on center.
In some embodiments as disclosed herein, a feature is about 0.5 micron to about 10 microns in diameter; about 11 microns to about 20 microns in diameter; about 21 microns to about 30 microns in diameter; about 31 microns to about 40 microns in diameter; about 41 microns to about 50 microns in diameter; about 51 microns to about 60 microns in diameter; about 61 microns to about 70 microns in diameter; about 71 microns to about 80 microns in diameter; about 81 microns to about 90 microns in diameter; about 91 microns to about 100 microns in diameter; about 101 microns to about 110 microns in diameter; about 111 microns to about 120 microns in diameter; about 121 microns to about 130 microns in diameter; about 131 microns to about 140 microns in diameter; about 141 microns to about 150 microns in diameter; about 151 microns to about 160 microns in diameter; about 161 microns to about 170 microns in diameter; about 171 microns to about 180 microns in diameter; about 181 microns to about 190 microns in diameter; about 191 microns to about 200 microns in diameter.
In some embodiments as disclosed herein, the center-to-center distance between adjacent features is about 1 micron to about 10 microns; about 11 microns to about 20 microns; about 21 microns to about 30 microns; about 31 microns to about 40 microns; about 41 microns to about 50 microns; about 51 microns to about 60 microns; about 61 microns to about 70 microns; about 71 microns to about 80 microns; about 81 microns to about 90 microns; about 91 microns to about 100 microns; about 101 microns to about 110 microns; about 111 microns to about 120 microns; about 121 microns to about 130 microns; about 131 microns to about 140 microns; about 141 microns to about 150 microns; about 151 microns to about 160 microns; about 161 microns to about 170 microns; about 171 microns to about 180 microns; about 181 microns to about 190 microns; about 191 microns to about 200 microns; about 201 microns to about 210 microns; about 211 microns to about 220 microns; about 221 microns to about 230 microns; about 231 microns to about 240 microns; about 241 microns to about 250 microns; about 251 microns to about 260 microns; about 261 microns to about 270 microns; about 271 microns about 280 microns; about 281 microns to about 290 microns; about 291 microns to about 300 microns.
In some embodiments as disclosed herein, the array comprises at least 10,000 features, at least 20,000 features, at least 30,000 features, at least 40,000 features, at least 50,000 features, at least 60,000 features, at least 70,000 features, at least 80,000 features, at least 90,000 features, at least 100,000 features, at least 110,000 features, at least 120,000 features, at least 130,000 features, at least 140,000 features, at least 150,000 features, at least 160,000 features, at least 170,000 features, at least 180,000 features, at least 190,000 features, at least 200,000 features, at least 210,000 features, at least 220,000 features, at least 230,000 features, at least 240,000 features, at least 250,000 features, at least 260,000 features, at least 270,000 features, at least 280,000 features, at least 290,000 features, at least 300,000 features, at least 310,000 features, at least 320,000 features, at least 330,000 features, at least 340,000 features, at least 350,000 features, at least 360,000 features, at least 370,000 features, at least 380,000 features, at least 390,000 features, at least 400,000 features, at least 410,000 features, at least 420,000 features, at least 430,000 features, at least 440,000 features, at least 450,000 features, at least 460,000 features, at least 470,000 features, at least 480,000 features, at least 490,000 features, at least 500,000 features, at least 600,000 features, at least 700,000 features, at least 800,000 features, at least 900,000 features, or at least 1,000,000 features.
In some embodiments as disclosed herein, the surface of the array is conditioned or pre-conditioned prior to attachment of the sequences onto the surface of the array. Conditioning of the array surface includes exposing the features on the surface to the coupling and photoresist solutions in the absence of monomers to form sequences.
In some embodiments as disclosed herein, at least one pre-conditioning or conditioning step is performed on the surface prior to the main synthesis of the functional sequence.
In some embodiments as disclosed herein, the conditioning step is performed at least 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 21 times, 22 times, 23 times, 24 times, 25 times, 26 times, 27 times, 28 times, 29 times, or 30 times.
In some embodiments as disclosed herein, the conditioning step is performed no more than 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 21 times, 22 times, 23 times, 24 times, 25 times, 26 times, 27 times, 28 times, 29 times, or 30 times.
In some embodiments as disclosed herein, the conditioning step is performed 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 21 times, 22 times, 23 times, 24 times, 25 times, 26 times, 27 times, 28 times, 29 times, or 30 times.
In some embodiments, conditioning the surface of the array as disclosed herein can increase the sensitivity of the array by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.
In some embodiments, conditioning the surface of the array as disclosed herein can increase the sensitivity of the array by at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or at least 15-fold.
In some embodiments, conditioning the surface of the array as disclosed herein can increase the specificity of the array, as compared to an array with a decreased length or shorter linker, by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.
In some embodiments, conditioning the surface of the array as disclosed herein can increase the specificity of the array, as compared to an array in the absence of conditioning, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or at least 15-fold.
In some embodiments, conditioning the surface of the array as disclosed herein can increase the reproducibility of the array, as compared to an array in the absence of conditioning, by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.
In some embodiments, conditioning the surface of the array as disclosed herein can increase the reproducibility of the array by at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or at least 15-fold.
In yet other embodiments, conditioning the surface of the array as disclosed herein can enhance binding of a target to the sequence on the array by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.
In still other embodiments, conditioning the surface of the array as disclosed herein can enhance binding of a target to the sequence on the array by at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or at least 15-fold.
Steps (1) through (4) describe the generation of an array with multiple successive additions of a specific epitope:
Step (1). An 8 inch silicon wafer with a 2500 angstrom thermal oxide layer was cleaned extensively with a solution of sulfuric acid (83.5% in volume) and 30% hydrogen peroxide (16.5% in volume). This surface was extensively flushed with highly purified water. The wafer was then placed in a 0.5% solution by volume of aminopropyltriethoxysilane in 95% ethanol and 4.5% water and allowed to sit without agitation for 90 minutes. The wafer was next washed in ethanol and isopropanol, spin-dried and placed in an oven at 110° C. for 90 minutes. The wafer was then stored under nitrogen for two days. Then the wafer was placed on a spin coater and a 10 mL coupling solution of Boc-Gly (0.1M), 6-Cl-HOBT (0.1M) and diisopropylcarbodiimide (DIC, 0.1M) in N-methylpyrrolidinone (NMP) was added while spinning at 500 RPM. The wafer was then removed from the spin coater and a second clean wafer was placed on top of it in such a way that the coupling solution spun onto it was trapped between the two wafers. This arrangement was placed on a hot plate at 85° C. for 120 seconds. The cover plate was removed, the wafer was placed back on the spin coater and 10 mL of NMP was added as the wafer was spun at 2000 RPM to wash off the coupling solution. This coupling was repeated one more time before it was washed on a spin coater with solvents in order of 10 mL N-methylpyrrolidinone (NMP), 10 mL methylisobutylketone (MIBK), and 10 mL isopropanol (IPA) at 2000 RPM.
Step (2). The wafer was then placed on a spin coater and 10 mL of a photoresist solution (25 mg/ml poly methyl methacrylate (PMMA), 30 mg/ml Bis(4-t-butylphenyl)iodonium triflate (PAG), and 15 mg/mL isopropyl-9H-thioxanthen-9-one (ITX) in propylene glycol methyl ether acetate (PGMEA)) were added while spinning at 2000 RPM. The wafer was then kept at 75° C. for 1.5 minutes. The wafer was placed on an aligner and aligned to a precise position relative to a mask (within approximately 1-2 microns in the two horizontal dimensions and 10 microns in the vertical dimension). The mask was designed to expose certain features on the wafer to UV light centered at 365 nm. The wafer was exposed through the mask for 7 seconds using a total exposure power of 19 milliwatts/cm2. The wafer was again placed on the spin coater, and 10 mL of methylisobutylketone (MIBK) was added while spinning at 2000 RPM to remove the photoresist. This was followed by 10 mL of isopropanol at the same speed. 10 mL of a coupling solution consisting of Boc-Ser (0.1M), DIC (0.1M), and 6-Cl-HOBT (0.1M) in NMP was then added while spinning at 500 RPM. The wafer was then removed from the spin coater and a second clean wafer was placed on top of it in such a way that the coupling solution spun onto it was trapped between the two wafers. This arrangement was placed on a hot plate at 85° C. for 120 seconds. The cover plate was removed, the wafer placed back on the spin coater and 10 mL of NMP was added as the wafer was spun at 2000 RPM to wash off the coupling solution. 10 mL of MIBK was then added as the wafer was spun at 2000 RPM, followed by 10 mL IPA at the same speed.
Step (3). The process described above in (2) was repeated 21 times, each time using an appropriate mask and amino acid. The amino acids were added in the following order: first=V; second=V; third=S; fourth=H; fifth=R; sixth=V; seventh=V; eight=S; ninth=H; tenth=R; eleventh=V; twelfth=V; thirteenth=S; fourteenth=H; fifteenth=R; sixteenth=V; seventeenth=V; eighteenth=S; nineteenth=H; twentieth=R; and each amino acid was added in same concentration of 0.1M as shown in (2). The one letter designation of each amino acid is used in the foregoing and that designation is well known to those skilled in the art.
Step (4). The wafer described above was diced in such a way that the arrays of ˜330,000 peptides could be individually exposed to specific samples. In this way arrays were incubated with 5 pM of a monoclonal antibody P53Ab1. There were some peptides with the sequence RHSVVGSG (SEQ ID NO: 7) on the array. In some cases the RHSVV (SEQ ID NO: 1) part of the peptide was synthesized early in the synthesis (designated RHSVVaGSG (SEQ ID NO: 7) in
The binding of the P53Ab1 monoclonal antibody to an array comprising each of the peptides described in EXAMPLE 1 was determined and is shown in
Step (1). An 8 inch silicon wafer with a 2,500 angstrom thermal oxide layer was cleaned extensively with a solution of sulfuric acid (83.5% in volume) and 30% hydrogen peroxide (16.5% in volume). This surface was extensively flushed with highly purified water. The wafer was then placed in a 0.5% solution by volume of aminopropyltriethoxysilane in 95% ethanol and 4.5% water and allowed to sit without agitation for 90 minutes. The wafer was next washed in ethanol and isopropanol and then placed in an oven at 110° C. for 90 minutes. The wafer was then stored under nitrogen for two days. Then the wafer was placed on a spin coater, and a 10 mL coupling solution of Boc-Gly (0.1M), 6-Cl-HOBT (0.1M), and diisopropylcarbodiimide (DIC) in N-methylpyrrolidinone (NMP) was added while spinning at 500 RPM. The wafer was then removed from the spin coater and a second clean wafer was placed on top of it in such a way that the coupling solution spun onto was trapped between the two wafers. This arrangement was placed on a hot plate at 85° C. for 120 seconds. The cover plate was removed, the wafer placed back on the spin coater and 10 ml of NMP was added as the wafer was spun at 2000 RPM to wash off the coupling solution. The coupling was repeated one more time before it was washed on a spin coater in order of 10 mL NMP, 10 mL MIBK, and 10 mL IPA, each at 2000 RPM.
Step (2) The wafer was then placed on a spin coater and 10 mL of a photoresist solution (25 mg/ml poly methyl methacrylate (PMMA), 30 mg/ml Bis(4-t-butylphenyl)iodonium triflate (PAG) and 15 mg/mL isopropyl-9H-thioxanthen-9-one (ITX) in propylene glycol methyl ether acetate (PGMEA)) were added while spinning at 2,000 RPM. The wafer was then placed on a hot place at 75° C. for 1.5 minutes. The wafer was placed on an aligner and aligned to a precise position relative to a mask (within approximately 1-2 microns in the two horizontal dimensions and 10 microns in the vertical dimensions). The mask was designed to expose certain features on the wafer to UV light centered at 365 nm. The wafer was exposed through the mask for 7 seconds using a total exposure power of 19 milliwatts/cm2. The wafer was again placed on the spin coater and 10 mL of methylisobutylketone was added while spinning at 2,000 RPM to remove the photoresist. This was followed by 10 mL of isopropanol at the same speed. 10 mL of a coupling solution consisting of Boc-Ser (0.1M), DIC (0.1M), and 6-Cl-HOBT (0.1M) in NMP was then added while spinning at 500 RPM. The wafer was then removed from the spin coater and a second clean wafer was placed on top of it in such a way that the coupling solution spun onto it was trapped between the two wafers. This arrangement was placed on a hot plate at 85° C. for 120 seconds. The cover plate was removed, the wafer placed back on the spin coater and 10 mL of NMP was added as the wafer was spun at 2,000 RPM to wash off the coupling solution. 10 mL of MIBK was then added as the wafer was spun at 2,000 RPM, followed by 10 mL IPA at the same speed to clean and dry the wafer.
Step (3) The process described above in (2) was repeated a number of times, each time using an appropriate mask and amino acid. The amino acids were added in the order: first=G; second=G; third=S; forth=G; fifth=G; sixth=S; seventh=G; eight=G; ninth=S; tenth=G; eleventh=G; twelfth=S; thirteenth=G; fourteenth=G; fifteenth=S; sixteenth=G; seventeenth=G; eighteenth=S; nineteenth=G; twentieth=G; twenty-first=S; twenty-second=G; twenty-third=V; twenty-fourth=V; twenty-fifth=S; twenty-six=H; twenty-seventh=R; twenty-eight=V; twenty-ninth=V; thirtieth=S; thirty-first=H; thirty-second=R; thirty-third=V; thirty-fourth=V; thirty-fifth=S; thirty-six=H; thirty-seventh=R; thirty-eighth=V; thirty-ninth=V; fortieth=S; fortieth-first=H; and fortieth-second=R. The masks were designed such that the number of G's and S's varied from one peptide to another, but all peptides ended with RHSVV (SEQ ID NO: 1) (the epitope for P53Ab1). All amino acids were added in the same concentration (0.1M) as shown in (2). The one letter designation of each amino acid is used in the foregoing and that designation is well known to those skilled in the art.
Step (4) The wafer described above was diced in such a way that the arrays of ˜330,000 peptides could be individually exposed to specific samples as described previously (Legutki et al., 2014, Nature Communications, 5, Article number: 4785 doi:10.1038/ncomms5785). In this way arrays were incubated with 5.0 pM of a monoclonal antibody P53Ab1.
Step (1). An 8 inch silicon wafer with a 2,500 angstrom thermal oxide layer was cleaned extensively with a solution of sulfuric acid (83.5% by volume) and 30% hydrogen peroxide (16.5% by volume). This surface was extensively flushed with highly purified water. The wafer was then placed in a 0.5% solution by volume of aminopropyltriethoxysilane in 95% ethanol and 4.5% water and allowed to sit without agitation for 90 minutes. The wafer was next washed in ethanol and isopropanol and placed in an oven at 110° C. for 90 minutes. Then the wafer was placed on a spin coater, and a 10 mL coupling solution of Boc-Gly (0.1M), 6-Cl-HOBT (0.1M), and diisopropylcarbodiimide (DIC, 0.1M) in N-methylpyrrolidinone (NMP) was added while spinning at 500 RPM. The wafer was then removed from the spin coater and a second clean wafer was placed on top of it in such a way that the coupling solution spun onto it was trapped between the two wafers. This arrangement was placed on a hot plate at 85° C. for 120 seconds. The cover plate was removed, the wafer placed back on the spin coater and 10 mL of NMP was added as the wafer was spun at 2,000 RPM to wash off the coupling solution. 10 mL of MIBK was then added as the wafer was spun at 2,000 RPM, followed by 10 mL IPA at the same speed to clean and dry the wafer. The coupling was repeated one more time.
Step (2). The wafer was placed on a spin coater and 10 mL of a photoresist solution (25 mg/ml poly methyl methacrylate (PMMA), 30 mg/ml Bis(4-t-butylphenyl)iodonium triflate (PAG) and 15 mg/mL isopropyl-9H-thioxanthen-9-one (ITX) in propylene glycol methyl ether acetate (PGMEA)) were added while spinning at 2,000 RPM. The wafer was then placed on a hot place at 75° C. for 1.5 minutes. The wafer was placed on an aligner and aligned to a precise position relative to a mask (within approximately 1-2 microns in the two horizontal dimensions and 10 microns in the vertical dimension). The mask was designed to expose certain features on the wafer is UV light centered at 365 nm. The wafer was exposed through the mask for 7 seconds using a total exposure power of 19 milliwatts/cm2. The wafer was again placed on the spin coater and 10 mL of methylisobutylketone was added while spinning at 2,000 RPM to remove the photoresist. This was followed by 10 mL of isopropanol at the same speed. 10 mL of a coupling solution consisting of Boc-Ser (0.1M), DIC (0.1M), and 6-Cl-HOBT (0.1M) in NMP was then added while spinning at 500 RPM. The wafer was then removed from the spin coater and a second clean wafer was placed on top of it in such a way that the coupling solution spun onto it was trapped between the two wafers. This arrangement was placed on a hot plate at 85° C. for 120 seconds. The cover plate was removed, the wafer placed back on the spin coater and 10 mL of NMP was added as the wafer was spun at 2,000 RPM to wash off the coupling solution. 10 mL of MIBK was then added as the wafer was spun at 2,000 RPM, followed by 10 mL IPA at the same speed to clean and dry the wafer.
Step (3). The process described above in (2) was then repeated 18 times except that the mask alignment and light exposure were removed from the process. These are the conditioning steps of the overall procedure but with only one amino acid, Boc-Gly, used in the concentration of 0.1M as shown in (2).
Step (4). The wafer was then placed on a spin coater and 10 mL of a photoresist solution (25 mg/ml poly methyl methacrylate (PMMA), 30 mg/ml Bis(4-t-butylphenyl)iodonium triflate (PAG) and 15 mg/mL isopropyl-9Hthioxanthen-9-one (ITX) in propylene glycol methyl ether acetate (PGMEA)) were added while spinning at 2,000 RPM. The wafer was then placed on a hot place at 75° C. for 1.5 minutes. The wafer was placed on an aligner and aligned to a precise position relative to a mask (within approximately 1-2 microns in the two horizontal dimensions and 10 microns in the vertical dimension). The mask was designed to expose certain features on the wafer to UV light centered at 365 nm. The wafer was exposed through the mask for 7 seconds using a total exposure power of 19 milliwatts/cm2. The wafer was again placed on the spin coater and 10 mL of MIBK was added while spinning at 2,000 RPM to remove the photoresist. This was followed by 10 mL of IPA at the same speed. 10 mL of a coupling solution consisting of Boc-Gly (0.1M), DIC (0.1M), and 6-Cl-HOBT (0.1M) in NMP was then added while spinning at 500 RPM. The wafer was then removed from the spin coater and a second clean wafer was placed on top of it in such a way that the coupling solution spun onto it was trapped between the two wafers. This arrangement was placed on a hot plate at 85° C. for 120 seconds. The cover plate was removed, the wafer placed back on the spin coater and 10 mL of NMP was added as the wafer was spun at 2,000 RPM to wash off the coupling solution. 10 mL of MIBK was then added as the wafer was spun at 2,000 RPM, followed by 10 mL IPA at the same speed.
Step (5). The process described above in (4) was repeated 69 times, each time using a different mask and a different amino acid. The amino acids were added in the order: first=G; second=G; third=G; fourth=R; fifth=L; sixth=Y; seventh=N; eight=H; ninth=S; tenth=D; eleventh=L; twelfth=W; thirteenth=K; fourteenth=E; fifteenth=H; sixteenth=K; seventeenth=W; eighteenth=D; nineteenth=V; twenty-first=Y; twenty-second=R; twenty-third=E; twenty-forth=F; twenty-fifth=P; twenty-sixth=V; twenty-seventh=F; twenty-eight=K; twenty-ninth; thirtieth=E; thirty-first=Q; thirty-second=N; thirty-third=Y; thirty-fourth=R; thirty-fifth=G; thirty-sixth=E; thirty-seventh=W; thirty-eight=D; thirty-ninth=G; fortieth=K; forty-first=Q; forty-second=L; forty-third; forty-fourth=Y; forty-fifth=V; forty-seventh=G; forty-eight=W; forty-ninth=E; fiftieth=G; fifty-first=S; fifty-second=P; fifty-third=R; fifty-fourth=V; fifty-fifth=P; fifty-sixth=H; fifty-seventh=R; fifty-eight=K; fifty-ninth=N; sixtieth=L; sixty-first=S; sixty-second=L; sixty-third=R; sixty-forth=A; sixty-fifth=D; sixty-eight=A; sixty-ninth=Y; seventieth=H; seventy-first=G; seventy-second=E; seventy-third=Q; seventy-forth=K; seventy-fifth=N). All amino acids were added in the same concentration of 0.1M. The one letter designation of each amino acid is used in the forgoing and that designation is well known to those skilled in the art.
Step (6). The wafer described above was diced such that arrays of ˜330,000 peptides could be individually exposed to specific samples as described previously. 24 blood samples from patients infected with Malaria, Dengue fever, West Nile Virus, or patients that were not infected were analyzed for binding patterns and the immunosignature was determined. A set of 50 peptides were chosen that provided the most unique antibody binding data for each of the diseases. See Legutki et al., Nat. Commun. 5:4785 (2014).
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
This application claims the benefit of U.S. Provisional Application No. 62/218,418, filed Sep. 14, 2015, which is incorporated herein by reference in its entirety.
This invention was made with government support under 1243082 awarded by the National Science Foundation. The government has certain rights in the invention.
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| Number | Date | Country | |
|---|---|---|---|
| 20170106344 A1 | Apr 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62218418 | Sep 2015 | US |
