Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
  • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
  • C08G73/02—Polyamines
  • C08G73/0206—Polyalkylene(poly)amines
• • C—CHEMISTRY; METALLURGY
  • C03—GLASS; MINERAL OR SLAG WOOL
  • C03C—CHEMICAL COMPOSITION OF GLASSES, GLAZES OR VITREOUS ENAMELS; SURFACE TREATMENT OF GLASS; SURFACE TREATMENT OF FIBRES OR FILAMENTS MADE FROM GLASS, MINERALS OR SLAGS; JOINING GLASS TO GLASS OR OTHER MATERIALS
  • C03C17/00—Surface treatment of glass, not in the form of fibres or filaments, by coating
  • C03C17/28—Surface treatment of glass, not in the form of fibres or filaments, by coating with organic material
  • C03C17/30—Surface treatment of glass, not in the form of fibres or filaments, by coating with organic material with silicon-containing compounds
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic System
  • C07F7/02—Silicon compounds
  • C07F7/08—Compounds having one or more C—Si linkages
  • C07F7/18—Compounds having one or more C—Si linkages as well as one or more C—O—Si linkages
  • C07F7/1804—Compounds having Si-O-C linkages
• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
  • C09D179/00—Coating compositions based on macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing nitrogen, with or without oxygen, or carbon only, not provided for in groups C09D161/00 - C09D177/00
  • C09D179/02—Polyamines
• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
  • C09D183/00—Coating compositions based on macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing silicon, with or without sulfur, nitrogen, oxygen, or carbon only; Coating compositions based on derivatives of such polymers
  • C09D183/04—Polysiloxanes
  • C09D183/08—Polysiloxanes containing silicon bound to organic groups containing atoms other than carbon, hydrogen, and oxygen
• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
  • C09D183/00—Coating compositions based on macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing silicon, with or without sulfur, nitrogen, oxygen, or carbon only; Coating compositions based on derivatives of such polymers
  • C09D183/14—Coating compositions based on macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing silicon, with or without sulfur, nitrogen, oxygen, or carbon only; Coating compositions based on derivatives of such polymers in which at least two but not all the silicon atoms are connected by linkages other than oxygen atoms
• • C—CHEMISTRY; METALLURGY
  • C03—GLASS; MINERAL OR SLAG WOOL
  • C03C—CHEMICAL COMPOSITION OF GLASSES, GLAZES OR VITREOUS ENAMELS; SURFACE TREATMENT OF GLASS; SURFACE TREATMENT OF FIBRES OR FILAMENTS MADE FROM GLASS, MINERALS OR SLAGS; JOINING GLASS TO GLASS OR OTHER MATERIALS
  • C03C2218/00—Methods for coating glass
  • C03C2218/10—Deposition methods
  • C03C2218/11—Deposition methods from solutions or suspensions
  • C03C2218/113—Deposition methods from solutions or suspensions by sol-gel processes
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T428/00—Stock material or miscellaneous articles
  • Y10T428/31504—Composite [nonstructural laminate]
  • Y10T428/31652—Of asbestos
  • Y10T428/31663—As siloxane, silicone or silane

Definitions

• This application relates to silicon compounds, methods of making silicon compounds, and methods for use of silicon compounds as silylating agents in the treatment of surfaces, such as glass.
• Silylating agents have been developed in the art which react with and coat surfaces, such as silica surfaces.
• silylating agents for use in modifying silica used in high performance chromatography packings have been developed.
• Monofunctional silylating agents have been used to form monolayer surface coatings, while di- and tri- functional silylating agents have been used to form polymerized coatings on silica surfaces.
• Many silylating agents produce coatings with undesirable properties including instability to hydrolysis and the inadequate ability to mask the silica surface which may contain residual acidic silanols.
• Silylating agents have been developed for the silylation of solid substrates, such as glass substrates, that include functional groups that may be derivatized by further covalent reaction.
• the silylating agents have been immobilized on the surface of substrates, such as glass, and used to prepare high density immobilized oligonucleotide probe arrays.
• substrates such as glass
• N-(3-(triethoxysilyl)-propyl)-4-hydroxybutyramide PCR Inc., Gainesville, FL and Gelest, Tullytown, PA
• PCR Inc. has been used to silylate a glass substrate prior to photochemical synthesis of arrays of oligonucleotides on the substrate, as described in McGall et al, J. Am. Chem. Soc, 119:5081-5090 (1997), the disclosure of which is incorporated herein by reference.
• hydroxyalkylsilyl compounds that have been used to prepare hydroxyalkylated substances, such as glass substrates.
• N,N-bis(hydroxyethyl) aminopropyl-triethoxysilane has been used to treat glass substrates to permit the synthesis of high-density oligonucleotide arrays. McGall et al., Proc. Natl. Acad. Sci., 93:13555-13560 (1996); and Pease et al, Proc. Natl. Acad. Sci, 91:5022-5026 (1994), the disclosures of which are incorporated herein.
• Acetoxypropyl-triethoxysilane has been used to treat glass substrates to prepare them for oligonucleotide array synthesis, as described in PCT WO 97/39151, the disclosure of which is incorporated herein.
• 3-Glycidoxy propyltrimethoxysilane has been used to treat a glass support to provide a linker for the synthesis of oligonucleotides.
• the functionalized silicon compounds include an activated silicon group and a derivatizable functional group.
• exemplary derivatizable functional groups include hydroxyl, amino, carboxyl and thiol, as well as modified forms thereof, such as activated or protected forms.
• the functionalized silicon compounds may be covalently attached to surfaces to form functionalized surfaces which may be used in a wide range of different applications.
• the silicon compounds are attached to the surface of a substrate comprising silica, such as a glass substrate, to provide a functionalized surface on the silica containing substrate, to which molecules, including polypeptides and nucleic acids, may be attached.
• an array of nucleic acids may be covalently attached to the substrate.
• the method permits the formation of high density arrays of nucleic acids immobilized on a substrate, which may be used in conducting high volume nucleic acid hybridization assays.
• Figure 1 shows the structure of the functionalized silicon compounds VI and VII and compounds of Formula 6a.
• Figure 2 shows the structure of the functionalized silicon compound VIII.
• Figure 3 shows schemes for the synthesis of compounds IX and X.
• Figure 4 is a scheme showing the synthesis of compounds of Formula 5 or 6.
• Figure 5 show schemes for the synthesis of compounds XII, XIII and compounds of Formula 8.
• Figure 6 shows schemes for the synthesis of compounds XV, VI and compounds of
• Figure 7 shows schemes showing the synthesis of compounds of Formula 10 or 11.
• Figure 8 shows schemes showing the synthesis of compounds of Formula 12 or 13.
• Figure 9 is a scheme of the synthesis of compounds of Formula 16b.
• Figure 10 illustrates the structure of compounds of Formula 14 and 15.
• Figure 11 is a graph of stability of silicon compound bonded phases vs. time.
• Figure 12 is a graph of hybridization fluorescence intensity vs. silane.
• Figure 13 is a scheme showing the synthesis of silicon compounds XVIa-e.
• Figure 14 is a scheme showing the synthesis of silicon compounds XVIIa-f.
• Figure 15 shows the structure of compounds of the general Formulas 17-21.
• Figure 16 shows the structure of some exemplary silicon compounds.
• Figure 17 shows another embodiment of exemplary silicon compounds XXI and XXII.
• Figure 18 shows the structure of exemplary silicon compounds XXIII, XXV and XXVI.
• Figure 19 shows the structure of exemplary silicon compounds XXIX and XXX.
• Figure 20 shows the structure of exemplary silicon compounds XXIa-b, XXIIa-b and XXIIIa-b.
• Figure 21 is a scheme showing the synthesis of silicon compounds XIX and XX.
• Figure 22 is a scheme showing the synthesis of silicon compounds XXI and XXII.
• Figure 23 is a scheme showing the synthesis of silicon compound XXIII.
• Figure 24 is a scheme showing the synthesis of silicon compound XXIV.
• Figure 25 is a scheme showing the synthesis of silicon compound XXVIII.
• Figure 26 is a scheme showing the synthesis of silicon compounds XXVI and XXV.
• Figure 27 is a scheme showing the synthesis of silicon compounds XXIX and XXX.
• Figure 28 is a scheme showing the synthesis of silicon compounds XXXIa-b and XXXIIIa-b.
• Figure 29 is a scheme showing the synthesis of silicon compounds XXXIIa-b.
• Figure 30 is a graph of normalized intensity vs. silane for silicon compounds bound to a solid substrate.
• Figure 31 is a graph of normalized hybridization fluorescence intensity vs. silane.
• Functionalized silicon compounds are provided, as well as methods for their synthesis and use.
• the functionalized silicon compounds may be used to form functionalized coatings on a variety of surfaces such as the surfaces of glass substrates.
• the functionalized silicon compounds may be used in the methods disclosed herein to react with surfaces to form functionalized surfaces which may be used in a wide range of different applications.
• the functionalized silicon compounds are covalently attached to surfaces to produce functionalized surfaces on substrates.
• the silicon compounds may be attached to the surfaces of glass substrates, to provide a functionalized surface to which molecules, including polypeptides and nucleic acids, may be attached.
• the term "silicon compound” refers to a compound comprising a silicon atom.
• the silicon compound is a silylating agent comprising an activated silicon group, wherein the activated silicon group comprises a silicon atom covalently linked to at least one reactive group, such as an alkoxy or halide, such that the silicon group is capable of reacting with a functional group, for example on a surface of a substrate, to form a covalent bond with the surface.
• the activated silicon group on the silicon compound can react with the surface of a silica substrate comprising surface Si-OH groups to create siloxane bonds between the silicon compound and the silica substrate.
• activated silicon groups include -Si(OMe) 3 ; - SiMe(OMe) 2 ; -SiMeCl 2 ; SiMe(OEt) 2 ; SiCl 3 and -Si(OEt) 3.
• the term "functionalized silicon compound” refers to a silicon compound comprising a silicon atom and a derivatizable functional group.
• the functionalized silicon compound is a functionalized silylating agent and includes an activated silicon group and a derivatizable functional group.
• the term "derivatizable functional group” refers to a functional group that is capable of reacting to permit the formation of a covalent bond between the silicon compound and another substance, such as a polymer.
• exemplary derivatizable functional groups include hydroxyl, amino, carboxy, thiol, and amide, as well as modified forms thereof, such as activated or protected forms.
• Derivatizable functional groups also include substitutable leaving groups such as halo or sulfonate.
• the derivatizable functional group is a group, such as a hydroxyl group, that is capable of reacting with activated nucleotides to permit nucleic acid synthesis.
• the functionalized silicon compound may be covalently attached to the surface of a substrate, such as glass, and then derivatizable hydroxyl groups on the silicon compound may be reacted with an activated phosphate group on a protected nucleotide phosphoramidite or H-phosphonate, and then stepwise addition of further protected nucleotide phosphoramidites or H- phosphonates can result in the formation of a nucleic acid covalently attached to the support.
• the nucleic acids also may be attached to the derivatizable group via a linker.
• arrays of nucleic acids may be formed covalently attached to the substrate which are useful in conducting nucleic acid hybridization assays.
• polynucleotide or “nucleic acid” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, that comprise purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
• the backbone of the polynucleotide can comprise sugars and phosphate groups, as may typically be found in RNA or DNA, or modified or substituted sugar or phosphate groups.
• a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
• the sequence of nucleotides may be interrupted by non-nucleotide components.
• the functionalized silicon compounds used to form coatings on a surface may be selected, and obtained commercially, or made synthetically, depending on their properties under the conditions of intended use.
• functionalized silicon compounds may be selected for silanization of a substrate that are stable after the silylation reaction to hydrolysis.
• the functionalized silicon compounds are used to form a coating on a solid substrate, and include functional groups that permit the covalent attachment or synthesis of nucleic acid arrays to the solid substrate, such as glass.
• the resulting substrates are useful in nucleic acid hybridization assays, which are conducted, for example in aqueous buffers.
• the functionalized surfaces on the substrate, formed by covalent attachment of functionalized silicon compounds advantageously are substantially stable to provide a support for biomolecule array synthesis and to be used under rigorous assay conditions, such as nucleic acid hybridization assay conditions.
• the functionalized silicon compound in one embodiment includes at least one activated silicon group and at least one derivatizable functional group.
• the functionalized silicon compound includes at least one activated silicon group and a plurality of derivatizable functional groups, for example, 2, 3, 4 or more derivatizable functional groups.
• the functionalized silicon compound includes at least one derivatizable functional group and a plurality of activated silicon groups, for example, 2, 3, 4 or more activated silicon groups.
• Methods of making the functionalized silicon compounds are provided as disclosed herein, as well as methods of use of the functionalized silicon compounds, including covalent attachment of the silicon compounds to surfaces of substrates to form functionalized surfaces, and further derivation of the surfaces to provide arrays of nucleic acids for use in assays on the surfaces.
• a method of functionalizing a surface comprising covalently attaching to the surface a functionalized silicon compound, wherein the functionalized silicon compound comprises at least one derivatizable functional group and a plurality of activated silicon groups, for example, 2, 3, 4 or more activated silicon groups.
• the method may further comprise covalently attaching a plurality of functionalized silicon compounds to the surface, and forming an array of nucleic acids covalently attached to the functionalized silicon compounds on the surface.
• Exemplary functionalized silicon compounds include compounds of Formula 1 shown below:
• Ri and R 2 are independently a reactive group, such as halide or alkoxy, for example -OCH 3 or -OCH 2 CH ; and R 3 is alkoxy, halide or alkyl; and wherein R 4 is a hydrophobic and/or sterically hindered group.
• R 4 may be alkyl or haloalkyl, for example, -CH 3 ,
• a hydrophobic and/or sterically hindered R group such as isopropyl or isobutyl, may be used to increase the hydrolytic stability of the resulting surface layer. Further hydrophobicity may be imparted by the use of a fluorocarbon R group, such as hexafluoroisopropyl ((CF 3 ) 2 CH-).
• An exemplary compound of Formula 1 is silicon compound I below:
• silicon compounds provide uniform and reproducible coatings.
• Silicon compounds with one derivatizable functional group can provide a lower concentration of surface derivatizable functional groups at maximum coverage of the substrate than the silicon compounds including multiple derivatizable functional groups.
• Ri, R 2 and R 3 are independently a reactive group, such as alkoxy or halide, for example, -OCH 3 , or -
• Ri, R 2 and R 3 are each -OCH 3.
• Ri and R 2 are independently a reactive group, such as alkoxy or halide, for example -OCH 3 or -OCH CH 3
• R 3 is an alkoxy or halide group or an alkyl group, such as -CH 3 , or substituted alkyl group.
• Ai is H or a moiety comprising one or more derivatizable functional groups.
• Ai is a moiety comprising an amino group or a hydroxyl group, such as -
• Ai is, for example, a branched hydrocarbon including a plurality of derivatizable functional groups, such as hydroxyl groups.
• A] is:
• Exemplary compounds of Formula 2 include compounds II, III and IV below.
• Other silicon compounds of Formula 2 that may be used to form functional surface coatings with enhanced hydrolytic stability include silicon compounds IX and X, shown in Figure 3.
• the triethoxysilyl group is shown by way of example, however alternatively, the activated silicon group may be other activated silicon groups or mixtures thereof, such as trimethoxysilyl.
• n is, for example, 1 to 10, e.g., 1-3
• G is a derivatizable functional group, such as hydroxyl, protected hydroxyl or halide such as Cl or Br, as shown in Figure 7.
• silicon compounds of Formula 14 in Figure 10 wherein, Rj, R are independently a reactive group such as alkoxy, for example -OCH 3 or - OCH CH 3 , or halide; and R 3 is a reactive group such as alkoxy or halide, or optionally alkyl.
• Rj, R are independently a reactive group such as alkoxy, for example -OCH 3 or - OCH CH 3 , or halide; and R 3 is a reactive group such as alkoxy or halide, or optionally alkyl.
• Ri, R , R 3 are independently a reactive group, such as alkoxy or halide, for example, -OCH 3 , or -OCH CH 3 , for example, in one embodiment, Ri, R 2 and R 3 are each -OCH 3 ; or in another embodiment, Ri and R are independently a reactive group, such as alkoxy or halide, for example -OCH 3 or -OCH 2 CH 3 , and R 3 is an alkoxy or halide group or an alkyl group, such as -CH 3 , or substituted alkyl group.
• Li and L may optionally comprise a heteroalkyl comprising a heteroatom such as O, S, or N.
• Each L] and L 2 independently comprise one or more derivatizable groups, e.g., 1-4 derivatizable groups, such as hydroxyl, amino or amido.
• Ai is H or a moiety comprising one or more derivatizable functional groups.
• a ⁇ is a moiety comprising an amino group or a hydroxyl group, such as -CH CH 2 OH.
• Ai is, for example, a linear or branched alkyl or heteroalkyl group including a plurality of derivatizable functional groups, for example, 1 , 2, or 3 derivatizable groups.
• A] may comprise a linear or branched alkyl or heteroalkyl, wherein one or more carbon atoms of the alkyl group is functionalized, for example, to comprise an amide.
• Examples of compounds include compounds XVIa-e shown in Figure 13, and compounds XVIIIa-f shown in Figure 14. Other examples include compound XII in Figure 5 and compound XV in Figure 6, as well as compounds XXIX and XXX in Figure 19.
• Ri, R , R 3 are independently a reactive group, such as alkoxy or halide, for example, -OCH 3 , or -
• Rj, R 2 and R 3 are each -OCH 3 ; or in another embodiment, R] and R 2 are independently a reactive group, such as alkoxy or halide, for example -OCH 3 or -OCH 2 CH 3 , and R 3 is an alkoxy or halide group or an alkyl group, such as -CH 3 , or substituted alkyl group.
• Lj, L 2 , and L 3 may optionally comprise a heteroalkyl comprising a heteroatom such as O, S, or N.
• Each Li, L , and L 3 independently optionally comprise one or more derivatizable groups, e.g., 1-4 derivatizable groups, such as hydroxyl or an amino group.
• Ai and A 2 may independently comprise H or a moiety comprising one or more derivatizable functional groups.
• Ai and A are independently moieties comprising an amino group or a hydroxyl group, such as -CH 2 CH 2 OH.
• Aj and A 2 may independently comprise, for example, a linear or branched alkyl or heteroalkyl including a plurality of derivatizable functional groups, for example, 1, 2, or 3 derivatizable groups.
• Ai and A may independently comprise a linear or branched alkyl or heteroalkyl, wherein one or more carbon atoms of the alkyl group is functionalized, for example, to an amide.
• Bi and B 2 are independently a branching group, for example alkyl, a heteroatom, or heteroalkyl, for example a Cl-12 alkyl.
• L 4 is a direct bond or a linker, for example, Cl-12 alkyl or heteroalkyl optionally comprising one more derivatizable groups.
• Examples of compounds of Formula 17 include compounds XIX, XX and XXIV in Figure 16.
• Examples of compounds of Formula 19 include compounds XXI and XXII in Figure 17.
• Other examples of compounds include compounds XXVII and XXVIII in Figure 16.
• compounds of Formula 18 include compounds XXXIa and XXXIb shown in Figure 20.
• Compounds of Formula 20 include compounds XXXIIa and XXXIIb shown in Figure 20.
• the compounds may include a single silicon group, as for example compounds XXXIIIa and XXXIIIb shown in Figure 20.
• Ri, R 2 , R 3 are independently a reactive group, such as alkoxy or halide, for example, -OCH 3 , or -OCH CH 3 , for example, in one embodiment, Ri, R 2 and R3 are each -OCH 3 ; or in another embodiment, R ⁇ and R 2 are independently a reactive group, such as alkoxy or halide, for example -OCH 3 or -OCH 2 CH 3 , and R 3 is an alkoxy or halide group or an alkyl group, such as -CH 3 , or substituted alkyl group.
• Li, L 2 , L 3 and L 4 may optionally comprise a heteroalkyl comprising a heteroatom such as O, S, or N.
• Each Li, L , L 3 and L independently optionally comprise one or more derivatizable groups, e.g., 1-4 derivatizable groups, such as hydroxyl or an amino group.
• Bi is a branching group, for example alkyl, a heteroatom, or heteroalkyl, for example a Cl-12 alkyl.
• Ai, A 2 , A 3 , and A may independently comprise H or a moiety comprising one or more derivatizable functional groups.
• a 1 ⁇ A 2 , A 3 , and A 4 are independently a moiety comprising an amino group or a hydroxyl group, such as -CH 2 CH OH.
• Aj, A 2 , A 3 , and A 4 may independently comprise, for example, an alkyl, such as a linear or branched alkyl, including a plurality of derivatizable functional groups, for example, 1, 2 or 3 derivatizable groups.
• A], A 2 , A 3 , and A 4 may independently comprise a linear or branched alkyl or heteroalkyl, wherein one or more carbon atoms of the alkyl group is functionalized, for example, to comprise an amide.
• Embodiments of compounds of Formula 21 include compounds XXIII, XXV and XXVI shown in Figure 18.
• Ri, R and R 3 are independently reactive groups, such as alkoxy or halide, for example, -OCH 3 , or -OCH 2 CH 3, and wherein, in one embodiment, Ri, R 2 and R 3 are each -OCH 3 .
• Ri and R are independently a reactive group, such as alkoxy or halide, for example -OCH 3 or -OCH CH 3
• R 3 is an alkoxy or halide group or an alkyl group, such as -CH 3 , or substituted alkyl group.
• A) and A are independently H or moieties comprising one or more derivatizable functional groups, such as hydroxyl or amino groups, or modified forms thereof, such as protected forms.
• a ⁇ and A each comprise a plurality of derivativizable functional groups.
• A[ and A 2 may each comprise a branched moiety including a plurality of derivatizable functional groups, such as hydroxyl groups.
• Ri and R 2 are independently alkoxy or halide; R 3 is alkoxy, halide or alkyl; Li , L 2 , and L 3 are independently -(CH 2 ) n -, wherein n is 2-10; and Ai and A 2 are independently a moiety comprising one or more derivatizable functional groups.
• A] is -L 4 -G ⁇ and A 2 is -L 5 -G ;
• Ri and R 2 are independently alkoxy or halide;
• R 3 is alkoxy, halide or alkyl;
• Lj, L 2 , L 3 , L 4 and L 5 are -
• (CH ) n - wherein n is 1 to 10, for example 2 to 3; and Gi and G are independently a moiety comprising one or more derivatizable functional groups.
• Lj, L 4 , and L 5 are -(CH 2 ) 2 -, L and L 3 are -(CH 2 ) 3 -, and Gi and G are -OH.
• silicon compounds of Formula 15 in Figure 10 are provided, wherein Ri, R 2 are independently alkoxy, for example -OCH 3 or -OCH CH 3 , or halide; and R 3 is alkoxy, alkyl, or halide.
• compounds of Formula 6a in Figure 1 are provided, wherein n is 1-3, for example 2 or 3.
• Exemplary functionalized silicon compounds include compound V below, and compound VI shown in Figure 1.
• silicon compounds II - V include two activated silicon groups for binding to a support surface, such as glass.
• a variety of functionalized silicon compounds including a plurality of activated silicon groups and derivatizable functional groups are useful to form functionalized coatings.
• a further example is compound VII shown in Figure 1.
• Another example is silicon compound VIII shown in Figure 2, which can form up to three covalent bonds to the surface of a glass support.
• the triethoxysilyl group is shown by way of example, however alternatively, the activated silicon group may be other activated silicon groups or mixtures thereof, such as trimethoxysilyl.
• the silicon compounds II-VIII having multiple silicon groups enhance potentially by twice as much, or more, the hydrolytic stability in comparison to silicon compounds comprising only a single silicon group, since they possess more trialkoxysilyl groups that can react, and form bonds with, a surface.
• the number of silicon groups in the silicon compound may be modified for different applications, to increase or decrease the number of bonds to a support such as a glass support.
• Silcon compounds may be used that form optimally stable surface-bonded films on glass via covalent siloxane bonds. Additionally, the number of derivatizable functional groups may be increased or decreased for different applications, as illustrated by silicon compounds II-VIII.
• Silicon compounds may be selected for use that provide the desired optimum density of surface derivatizable groups, such as hydroxyalkyl groups, for a desired application, such as the synthesis of nucleic acid arrays, or for the optimum stability during use of the array in different applications.
• Other embodiments of functionalized silicon compounds include compound XIII shown in Figure 5.
• polymeric functionalized silicon compounds of Formula 4 are provided:
• x, y and z are independently 1-3 and, in one embodiment, x, y and z are each 2.
• At least one of A, B and C is -SiR ⁇ R 2 R 3 , wherein Rj and R are independently a reactive group, such as alkoxy or halide, for example, -OCH 3 , or -OCH CH 3 and R 3 is alkoxy, halide or alkyl; and wherein the remainder of A, B and C are independently moieties comprising one or more derivatizable functional groups, such as hydroxyl groups, or amino groups, or modified forms thereof, such as protected forms, for example -OH or a branched molecule comprising one or more hydroxyl groups.
• n is, for example, about 10 to 10,000, or, for example, about 1,000 to 10,000.
• B is -SiR ⁇ R 2 R 3 , wherein Ri, R 2 and R 3 are independently alkoxy, halide or alkyl; x, y, and z are independently 2-3; Li, L 2 and L 3 are independently -(CH 2 ) m -, wherein m is 2-3; A and C are independently moieties comprising derivatizable functional groups; and n is about 10 to 10,000.
• B is -Si(OCH 3 ) 3 ; x, y, and z are 2; Li and L 3 are - (CH 2 ) 2 -; L 2 is -(CH 2 ) 3 -; A and C are moieties comprising derivatizable functional groups; and n is about 10 to 10,000.
• a polymeric functionalized silicon compound examples include compounds of Formula 5 and 6 shown in Figure 4, wherein m is about 0 to 10, e.g., about 1 to 5, and n is about 10 to 10,000.
• Ri and R 2 are independently a reactive group, such as alkoxy or halide, for example, -OCH 3 or -OCH 2 CH 3
• R 3 is a reactive group, such as alkoxy or halide, or optionally alkyl, for example -CH 3 .
• Rj and R are independently a reactive group, such as alkoxy or halide, for example, -OCH 3 or -OCH 2 CH 3
• R 3 is a reactive group such as alkoxy or halide or optionally alkyl, for example -CH 3 .
• Rj and R 2 are independently a reactive group, such as alkoxy or halide, for example, -OCH 3 or -OCH 2 CH 3
• R 3 is a reactive group, such as alkoxy or halide, or optionally alkyl, for example -CH 3
• G is a substitutable leaving group, such as hydroxy, protected hydroxy, or halo, such as -Cl or -Br.
• silicon compounds Commercially available silicon compounds and a review of silicon compounds is provided in Arkles, Ed., "Silicon, Germanium, Tin and Lead Compounds, Metal Alkoxides, Diketonates and Carboxylates, A Survey of Properties and Chemistry," Gelest, Inc., Tullytown, PA, 1995, the disclosure of which is incorporated herein.
• Functionalized silicon compounds may be synthesized using methods available in the art of organic chemistry, for example, as described in March, Advanced Organic Chemistry, John Wiley & Sons, New York, 1985, and in R.C. Larock, Comprehensive Organic Transformations, Wiley- VCH, New York, 1989.
• Scheme II A method for the conversion of compound XI, bis [3 -trimethoxysilylpropyl] - ethylenediamine, which is commercially available from Gelest, Inc., Tullytown, PA, to compound V is shown below in Scheme III.
• Reaction schemes for the synthesis of functionalized silicon compounds IX and X are provided in Figure 3. Reaction schemes for the synthesis of compounds of Formulas 5 and 6 are shown in Figure 4. Polyethyleneimine is available commercially, for example, from Aldrich®. Polyamines of Formula 5, where Ri, R and R 3 are OMe (trimethoxysilylpropyl modified (polyethyleneimine)), or Ri is Me and R and R 3 are OMe (dimethoxymethylsilylpropyl modified (polyethylenimine)) are available from Gelest
• Figure 9 shows another embodiment of a reaction scheme using commercially available reagents, wherein the compound of Formula 16a is converted to the compound of Formula 16b. Reaction schemes for the synthesis of compounds XII, XIII, and compounds of
• Formula 8 are shown in Figure 5. Synthesis of the reagent, N,N-bis(2- hydroxyethyl)acrylamide is described in U.S. Patent No. 3,285,886 (1966), the disclosure of which is incorporated herein.
• Figure 7 illustrates reaction schemes for the synthesis of compounds of Formulas 10 and 11.
• the use of the reagent, 4-chlorobutanoyl chloride, is described in Njoroge et al, PCT US97/15899 (1998), the disclosure of which is incorporated herein.
• Other reagents include lactones, such as ⁇ -butyrolactone, ⁇ -valerolactone, and ⁇ -caprolactone (Aldrich®).
• Figure 8 illustrates reaction schemes for compounds of Formulas 12 and 13.
• G is a substitutable leaving group such as halo.
• Figures 13 and 14 illustrate examples of methods of synthesis of compounds of Formula 2, compounds XVIa-e, and XVIIa-f.
• Figure 13 illustrates examples of methods of synthesis where a compound containing a primary amino function reacts with two equivalents of (3- glycidoxypropyl)trimethoxysilane (available from Gelest, Inc., Tullytown, PA) to provide tertiary amine containing compounds XVIa-XVIe.
• 3- glycidoxypropyl)trimethoxysilane available from Gelest, Inc., Tullytown, PA
• Figure 14 illustrates examples of methods of synthesis where a compound containing a primary amino function reacts with a carbamoyl chloride (produced by reaction of XIV with triphosgene or other phosgene synthon) to produce the substituted urea compounds XVIIa-XVIIf.
• Figure 21 provides an exemplary reaction scheme for compounds of Formula 17.
• the exemplary compounds XIX and XX are synthesized through the common intermediate 4-amino-l,6-heptadiene. This intermediate is prepared from ethylformimidate and methylmagnesium bromide (both available from Aldrich, Milwaukee, WI); the preparation is described in Barbot, F.; Tetrahedron Lett. 1989, 30, 185 and Barber, H. J.; J. Chem. Soc.
• the exemplary compounds XXI and XXII are synthesized through the common intermediate 4-allyl-4-amino-l,6-heptadiene.
• This intermediate can be prepared, for example, from ethylformimidate and methylmagnesium bromide (both available from Aldrich, Milwaukee, WI); the preparation is described in Barbot, F., Tetrahedron Lett. 1989, 30, 185; and Barber, H. J., J. Chem. Soc. 1943, 101.
• the intermediate also may be prepared from triallylborane as described in Bubnov, Y. N., et al., Russian Chem. Bull. 1996, 45, 2598.
• Figure 23 provides an exemplary reaction scheme for compounds of Formula 21, including structure XXIII.
• This compound is prepared by catalytic hydrogen reduction of 4,4-dicyano-l,6-bis(triethoxysilyl)heptane followed by reaction of the resultant diamine with ethylene oxide.
• the dicyano intermediate is prepared, for example, by a malonitrile synthesis of 4,4-dicyano-l,6-heptadiene followed by hydrosilylation, or by alkylation of malonitrile with 2 equivalents of a 3-halo-l-(triethoxysilyl)propane compound.
• Figure 24 provides an exemplary reaction scheme for a compound of Formula 17, including structure XXIV.
• This compound is prepared by catalytic hydrogen reduction of 4-cyano-l,6-bis(triethoxysilyl)heptane followed by reaction of the resultant amine with ethylene oxide.
• the cyano intermediate is prepared starting from a malonitrile synthesis of
• Figure 25 provides an exemplary reaction scheme for compounds of Formula 19, for example, structures XXVII and XXVIII. These compounds are prepared by reaction of appropriate primary (XXVIII) or secondary (XXVII) amines with ethyl 1 ,7- bis(triethoxysilyl)-4-heptanoate.
• the ester precursor is synthesized starting from diethyl 2,2-diallylmalonate (Aldrich, Milwaukee, WI); the diester is decarboxylated following the method of Beckwith, A. C. J.; et al, J. Chem. Soc, Perkin Trans. II, 1975, 1726, to produce ethyl 4-hepta-l,6-dieneoate; the triethoxysilyl moieties are introduced by hydrosilylation.
• Figure 26 provides an exemplary reaction scheme for compounds of Formula 21, for example, structures XXV and XXVI. These compounds are prepared by reaction of appropriate primary (XXVI) or secondary (XXV) amines with diethyl 2,2-bis(3- triefhoxysilylpropyl)malonate.
• the diester precursor can be synthesized by hydrosilylation of diethyl 2,2-diallylmalonate (Aldrich, Milwaukee, WI).
• FIG. 27 Another embodiment of a synthesis of a compound of Formula 2 is shown in Figure 27, wherein the synthesis of compounds XXIX and XXX is shown.
• the general method illustrated here is to N-alkylate (XXX) or N-acylate (XXIX) bis(3-trimethoxypropyl)amine (compound XI, Gelest, Inc., Tullytown, PA) with a alkyl chain containing an ester of a carboxylic acid that can be subjected to aminolysis with dihydroxyethylamine (Aldrich,
• alkylating agent methyl 4-chlorobutyrate
• acylating agent methyl 4-chloro-4-oxobutyrate
• Figure 28 provides an exemplary reaction scheme for compounds of Formula 18, for example, structures XXXIa and XXXIb. Preparation begins with the formation of 1,6- heptadiene-4-ol from reaction of ethyl formate with allylmagnesium bromide (Aldrich,
• Preparation begins with the formation of triallylmethanol from reaction of diethyl carbonate with allylmagnesium bromide by the method of Dreyfuss, M. P., J. Org. Chem. 1963, 28, 3269. Reaction of the alcohol with zinc chloride by the method of Reeve, W., J. Org. Chem. 1969, 34, 192 affords a halo diene which after hydrosilylation can be used to alkylate dihydroxyethylamine (Aldrich, Milwaukee, WI).
• XXXIIIb compounds which contain a single silicon atom. Preparation begins with the alkylation of dihydroxyethylamine (Aldrich, Milwaukee, WI) with an appropriate 3 -halo- 1- trialkoxylsilylproane reagent, followed by N-acylation with acroyl chloride (XXXIIIa) or methacroyl chloride (XXXIIIb) (Aldrich, Milwaukee, WI) following the procedure of Yokota, M., et al. , European patent Application 97309882.5, 1998 affords the desired compounds.
• Functionalized silicon compounds within the scope of the invention that may be used to form functionalized covalent coatings on surfaces that are useful in a variety of applications and assays further include amine compounds such as compound XI, as well as reaction products formed therefrom as disclosed herein.
• the methods and compositions disclosed herein may be used in a variety of applications.
• the functionalized silicon compounds may be covalently attached to a variety of materials, to provide derivatizable functional groups on the materials.
• Exemplary materials include materials that comprise a functional group that is capable of reacting with the activated silicon group of the silicon compound.
• the material may comprise a silica material comprising surface silanols capable of reacting with the activated silicon group to form a siloxane bond between the silicon atom on the silicon compound and the silicon atom on the surface.
• the functionalized silicon compounds may be attached to, for example, materials comprising silica, such as glass, chromatography material, and solid supports used for solid phase synthesis, such as nucleic acid synthesis.
• the functionalized silicon compounds further may be attached to materials comprising oxides such as titanium(IV) dioxide and zirconium dioxide, aluminum oxide and indium-tin oxides, as well as nitrides, such as silicon nitride.
• Solid substrates which may be coated by the silicon compounds include any of a variety of fixed organizational support matrices.
• the substrate is substantially planar.
• the substrate may be physically separated into regions, for example, with trenches, grooves, wells and the like.
• substrates include slides, beads and solid chips.
• the solid substrates may be, for example, biological, nonbiological, organic, inorganic, or a combination thereof, and may be in forms including particles, strands, gels, sheets, tubing, spheres, containers, capillaries, pads, slices, films, plates, and slides depending upon the intended use.
• the functionalized silicon compounds used advantageously may be selected with selected properties for a particular application.
• Functionalized silicon compounds may be selected which can form silicon compound surface coatings that have good stability to hydrolysis.
• Functionalized silicon compounds may be selected which have a selected reactivity with the substrate and a selected derivatizable functional group depending on the intended use.
• the functionalized silicon compounds may be covalently attached to the surface of a solid substrate to provide a coating comprising derivatizable functional groups on the substrate, thus permitting arrays of immobilized oligomers to be covalently attached to the substrate via covalent reaction with the derivatizable functional groups.
• the immobilized oligomers such as polypeptides, or nucleic acids can be used in a variety of binding assays including biological binding assays.
• high density arrays of immobilized nucleic acid probes may be formed on the substrate, and then one or more target nucleic acids comprising different target sequences may be screened for binding to the high density array of nucleic acid probes comprising a diversity of different potentially complementary probe sequences.
• the substrate may be, for example, silicon or glass, and can have the thickness of a microscope slide or glass cover slip. Substrates that are transparent to light are useful when the assay involves optical detection, as described, e.g., in U.S. Patent No. 5,545,531, the disclosure of which is incorporated herein.
• Other substrates include Langmuir Blodgett film, germanium, (poly)tetrafluorethylene, polystyrene, (poly)vinylidenedifluoride, polycarbonate, gallium arsenide, gallium phosphide, silicon oxide, silicon nitride, and combinations thereof.
• the substrate is a flat glass or single crystal silicon surface with relief features less than about
• the surfaces on the solid substrates will usually, but not always, be composed of the same material as the substrate.
• the surface may comprise any number of materials, including polymers, plastics, resins, polysaccharides, silica or silica based materials, carbon, metals, inorganic glasses, membranes, or any of the above-listed substrate materials.
• the surface will contain reactive groups, such as carboxyl, amino, and hydroxyl.
• the surface is optically transparent and will have surface Si-OH functionalities such as are found on silica surfaces.
• the number of nucleic acid sequences may be selected for different applications, and may o be, for example, about 100 or more, or, e.g., in some embodiments, more than 10 or 10 .
• the surface comprises at least 100 probe nucleic acids each preferably having a different sequence, each probe contained in an area of less than about 0.1 cm 2 , or, for example, between about 1 ⁇ m 2 and 10,000 ⁇ m 2 , and each probe nucleic acid having a defined sequence and location on the surface.
• each nucleic acid is contained within an area less than about 10 " cm , as described, for example, in U.S. Patent No. 5,510,270, the disclosure of which is incorporated herein.
• arrays of nucleic acids for use in gene expression monitoring are described in PCT WO 97/10365, the disclosure of which is incorporated herein.
• arrays of nucleic acid probes are immobilized on a surface, wherein the array comprises more than 100 different nucleic acids and wherein each different nucleic acid is localized in a predetermined area of the surface, and the density of the different nucleic acids is greater than about 60 different nucleic acids per 1 cm 2 .
• Arrays of nucleic acids immobilized on a surface which may be used also are described in detail in U.S. Patent No. 5,744,305, the disclosure of which is incorporated herein.
• nucleic acids with different sequences are immobilized each in a predefined area on a surface.
• 10, 50, 60, 100, 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , or 10 8 different monomer sequences may be provided on the substrate.
• the nucleic acids of a particular sequence are provided within a predefined region of a substrate, having a surface area, for example, of about 1 cm to 10 " cm . In some embodiments, the regions have areas of less than about 10 "1 , 10 "2 , 10 "3 , 10 "4 , 10 "5 , 10 "6 , 10 “7 , 10 “8 , 10 “9 , or 10 " '° cm 2 .
• a planar, non- porous support having at least a first surface, and a plurality of different nucleic acids attached to the first surface at a density exceeding about 400 different nucleic acids/cm , wherein each of the different nucleic acids is attached to the surface of the solid support in a different predefined region, has a different determinable sequence, and is, for example, at least 4 nucleotides in length.
• the nucleic acids may be, for example, about 4 to 20 nucleotides in length.
• the number of different nucleic acids may be, for example, 1000 or more.
• detection may be implemented by directing light to relatively small and precisely known locations on the substrate.
• the substrate is placed in a microscope detection apparatus for identification of locations where binding takes place.
• the microscope detection apparatus includes a monochromatic or polychromatic light source for directing light at the substrate, means for detecting fluoresced light from the substrate, and means for determining a location of the fluoresced light.
• the means for detecting light fluoresced on the substrate may in some embodiments include a photon counter.
• the means for determining a location of the fluoresced light may include an x/y translation table for the substrate. Translation of the substrate and data collection are recorded and managed by an appropriately programmed digital computer, as described in U.S. Patent No. 5,510,270, the disclosure of which is incorporated herein.
• Devices for concurrently processing multiple biological chip assays may be used as described in U.S. Patent No. 5,545,531, the disclosure of which is incorporated herein.
• Methods and systems for detecting a labeled marker on a sample on a solid support, wherein the labeled material emits radiation at a wavelength that is different from the excitation wavelength, which radiation is collected by collection optics and imaged onto a detector which generates an image of the sample are disclosed in U.S. Patent No. 5,578,832, the disclosure of which is incorporated herein. These methods permit a highly sensitive and resolved image to be obtained at high speed. Methods and apparatus for detection of fluorescently labeled materials are further described in U.S. Patent Nos.
• Arrays of polymers such as nucleic acids may be screened for specific binding to a target, such as a complementary nucleotide, for example, in screening studies for determination of binding affinity and in diagnostic assays.
• sequencing of polynucleotides can be conducted, as disclosed in U.S. Patent No. 5,547,839, the disclosure of which is incorporated herein.
• the nucleic acid arrays may be used in many other applications including detection of genetic diseases such as cystic fibrosis, diabetes, and acquired diseases such as cancer, as disclosed in U.S. Patent Application Ser. No. 08/143,312, the disclosure of which is incorporated herein.
• Genetic mutations may be detected by sequencing by hydridization.
• genetic markers may be sequenced and mapped using Type-IIs restriction endonucleases as disclosed in U.S. Patent No. 5,710,000, the disclosure of which is incorporated herein.
• Other applications include chip based genotyping, species identification and phenotypic characterization, as described in U.S. Patent Application Serial No. 08/797,812, filed February 7, 1997, and U.S. Application Serial No. 08/629,031, filed April 8, 1996, the disclosures of which are incorporated herein.
• Gene expression may be monitored by hybridization of large numbers of mRNAs in parallel using high density arrays of nucleic acids in cells, such as in microorganisms such as yeast, as described in Lockhart et al, Nature Biotechnology, 14:1675-1680 (1996), the disclosure of which is incorporated herein.
• Bacterial transcript imaging by hybridization of total RNA to nucleic acid arrays may be conducted as described in Saizieu et al, Nature Biotechnology, 16:45-48 (1998), the disclosure of which is incorporated herein. All publications cited herein are incorporated herein by reference in their entirety.
• Example 1 Silicon compounds were obtained commercially or synthesized from commercially available starting materials. Silicon compounds N,N-bis(2-hydroxyethyl)-3- aminopropyltriethoxysilane, and N-(2-hydroxyethyl)-N-methyl-3- aminopropyltriethoxysilane (compound I) were purchased from Gelest, Inc (Tullytown, PA). Silicon compounds II and V and were prepared as shown in Schemes II and III.
• Substrates were treated by a silanation procedure as follows. Glass substrates (borosilicate float glass, soda lime or fused silica, 2"x 3" x 0.027", obtained from U.S. Precision Glass (Santa Rosa, CA) were cleaned by soaking successively in Nanostrip
• Substrates were then spin-dried for 5 minutes under a stream of nitrogen at 35°C. Silanation was carried out by soaking under gentle agitation in a freshly prepared 1-2% (wt/vol) solution of the silicon compound in 95:5 ethanol-water for 15 minutes. The substrates were rinsed thoroughly with 2-propanol, then deionized water, and finally spin- dried for 5 minutes at 90°- 110°C.
• the stability of silicon compound bonded phase was evaluated.
• the surface hydroxyalkylsilane sites on the resulting substrates were "stained" with fluorescein in a checkerboard pattern by first coupling a MeNPOC-HEG linker phosphoramidite, image- wise photolysis of the surface, then coupling to the photo-deprotected linker sites a 1 :20 mixture of fluorescein phosphoramidite and DMT-T phosphoramidite (Amersham- Pharmacia Biotech, Piscataway, NJ), and then deprotecting the surface molecules in 1 :1 ethylenediamine-ethanol for 4 hr.
• the steps were conducted using standard protocols, as described in McGall et al, J. Am. Chem. Soc, 119: 5081-5090 (1997), the disclosure of which is incorporated herein.
• the pattern and intensity of surface fluorescence was imaged with a scanning laser confocal fluorescence microscope, which employed excitation with a 488 nm argon ion laser beam focused to a 2 micron spot size at the substrate surface. Emitted light was collected through confocal optics with a 530(+l 5) nm bandpass filter and detected with a
• Output intensity values are proportional to the amount of surface-bound fluorescein, so that relative yields of free hydroxyl groups within different regions of the substrate could be determined by direct comparison of the observed surface fluorescence intensities. All intensity values were corrected for nonspecific background fluorescence, taken as the surface fluorescence within the non-illuminated regions of the substrate.
• the relative surface reactive site density was measured. For each silicon compound tested, the number of available surface synthesis sites achieved per unit area was estimated, relative to N,N-bis(2-hydroxyethyl)-3-aminopropyl-triethoxysilane, by comparison of the observed initial surface fluorescence intensities of the various substrates immediately after deprotection in ethanolic diaminoethane.
• Site Density (% rel.) Intensity (silicon compound "X") x 100
• substrates were gently agitated on a rotary shaker at 45°C in 5xSSPE or 6xSSPE aqueous buffer
• a nucleic acid probe sequence (5'-GTC AAG ATG CTA CCG TTC AG-3') (SEQ. ID NO. 1) was synthesized photolithographically in a checkerboard array pattern (400 x 400 micron features) on the substrates that had been derivatized with either N,N-bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane or silicon compound V.
• the arrays were hybridized with a fluorescein-labeled complementary "target" nucleic acid (5'-fluorescein-CTG AAC GGT AGC ATC TTG AC-3') (SEQ. ID NO. 2) at a concentration of 250 pM in 6xSSPE buffer (0.9 M NaCl, 60 mM NaH 2 PO 4 , 6 mM EDTA, pH 7.5) for 16 hours at 45°C.
• 6xSSPE buffer 0.9 M NaCl, 60 mM NaH 2 PO 4 , 6 mM EDTA, pH 7.5
• the relative amount of bound target was determined from the fluorescence signal intensity.
• a graph of fluorescence signal intensity vs. silane is shown in Figure 12.
• Example 2 Silicon compound XVIb was prepared as shown in Figure 13. An oven dried 25 ml single-neck flask was charged with 1.07 g (1 ml, 0.0066 mol) N,N-bis(2 -hydroxy ethyl)- 1,3- diaminopropane (TCI America, Portland, OR) and 5 ml anhydrous methanol (Aldrich Chemical; Milwaukee, WI). To this stirring solution was added 2.92 ml (3.12 g, 0.0132 mol) (3-glycidoxypropyl)trimethoxysilane (Gelest, Inc., Tullytown, PA) dropwise over a 30 minute period.
• Silicon compound XVIIb was prepared as shown in Figure 14. An oven dried 25 ml single-neck flask was charged with 0.21 ml (0.15 g, 0.0015 mol) triethylamine (Aldrich Chemical; Milwaukee, WI), 0.61 g of a carbamoyl chloride prepared as per Example 5 (0.0015 mol), and 5 ml anhydrous THF (Aldrich Chemical; Milwaukee, WI). After addition of 0.20 g (0.0015 mol) diethanolamine (Aldrich Chemical; Milwaukee, WI) a precipitate formed. After 5 days of stirring under Ar at room temperature, the mixture was filtered and concentrated to a brown oil. After drying under vacuum, 0.62 g (87% crude yield) of material was obtained.
• Silicon compound XVIIf was prepared as shown in Figure 14. An oven dried 25 ml single-neck flask was charged with 42.4 ml (30.8 g, 0.304 mol) triethylamine (Aldrich Chemical; Milwaukee, WI), 116 g of a carbamoyl chloride prepared as per Example 5 (0.304 mol), and 500 ml anhydrous THF (Aldrich Chemical; Milwaukee, WI). After addition of 1.07 g (1 ml, 0.0066 mol) N,N-Bis(2-hydroxyethyl)-l,3-diaminopropane (TCI America, Portland, OR), 500 ml anhydrous CH 2 C1 (Aldrich Chemical; Milwaukee, WI) was added.
• Coated substrates with XVIb, XVIe, XVIIb, and XVIIf were prepared by a modified version of the silanation procedure of Example 1.
• the silane content of the 95:5 ethanokwater bath was 1-2% by volume of the methanol/silane compound solutions described in Examples 2-4 and 6-7 and curing was done at 50°C for 2 minutes.
• the surface was imaged after a period of 1 hour exposure to 5 nM target in 6X SSPE buffer (0.9 M NaCl, 60 mM NaH 2 PO 4 , 6 mM EDTA, pH 7.5) at 25°C and again after 16 hours at 45°C. Except as noted herein, the method employed for the assay is the same as described in Example 1.
• the fluorescence intensity was averaged and evaluated at both the 1 hour and 16 hour time points. Relative surface reactive site density was determined by normalization of the signal to that obtained from compound I as described in Example 1.
• Figure 30 shows the normalized data at the 1 and 16 hour time points. The percentage of remaining fluorescence intensity is noted for the 16-hour time point for each silane coating.

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