WO2003076903A2

WO2003076903A2 - Articles of manufacture and methods for making hydrophobic derivatized arrays

Info

Publication number: WO2003076903A2
Application number: PCT/US2003/007382
Authority: WO
Inventors: Shishir Shah; Jason Kang
Original assignee: Spectral Genomics, Inc.
Priority date: 2002-03-08
Filing date: 2003-03-10
Publication date: 2003-09-18
Also published as: WO2003076903A3; AU2003220159A8; AU2003220159A1

Abstract

The present invention provides articles of manufacture, e.g., arrays and devices, and methods for making and using them. The invention relates to techniques for immobilizing compositions, e.g., biological molecules, e.g., nucleic acids, to substrate surfaces for the purpose of conducting scientific investigations or routine testings. In particular, the invention provides articles of manufacture comprising covalently bound biological molecules on substrate surfaces that have been derivatized with a hydrophobic monolayer.

Description

ARTICLES OF MANUFACTURE AND METHODS FOR MAKING HYDROPHOBIC DERTVATIZED ARRAYS

Related Applications This application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 60/363,337, filed March 8, 2002. The aforementioned application is explicitly incorporated herein by reference in its entirety and for all purposes.

TECHNICAL FIELD

The present invention provides articles of manufacture, e.g., arrays and devices, and methods for making and using them. The invention relates to techniques for immobilizing compositions, e.g., biological molecules, such as nucleic acids or polypeptides, or small molecules, to substrate surfaces for the purpose of conducting scientific investigations or routine testing. The articles of manufacture of the invention can be used for genome-wide genetic mapping and gene expression studies, protein interaction studies, peptide interaction studies and small molecule interactions with larger macromolecules. In particular, the invention provides articles of manufacture comprising covalently bound compositions, e.g., biological molecules, on substrate surfaces that have been derivatized with a hydrophobic monolayer.

BACKGROUND To facilitate the study of a biological molecule such as a nucleic acid, it is often desirable to affix or immobilize it on a solid surface, such as a smooth sheet of glass. Fixed in place in this manner, a biological molecule can be readily manipulated or tested. For example, a biological molecule can be a long stranded polymer. Thus, immobilizing a polymeric molecule means fixing one end to a solid support such that the remainder of the polymer, or strand, is unmodified and firee to undergo further reaction (e.g., hybridization), depending upon the particular study. This is a widely used method to conduct laboratory studies involving DNA.

Perhaps the major problem associated with immobilizing a biological molecule on a solid support is exactly how to do it without altering the molecule other than that relatively small portion that is to be actually bound to the solid support. This is a very difficult problem because whatever solid support is used must be essentially inert. That is, it must not react with the biological molecule other than simply to immobilize it upon the solid support.

Glass is a particularly suitable solid support because it is inexpensive and highly inert. At present, the current orthodoxy is that the solid support (e.g., a glass surface) must first be primed or derivatized so that it can bind one end of the DNA to the surface. Numerous techniques exist to do this. Unfortunately, derivatizing the otherwise inert surface of glass creates problems that could confound the results of the laboratory study involving polymeric biological molecules. One problem is that derivatizing the glass surface may create a net positive electrostatic charge on the glass surface. Since many biological molecules, such as DNA, have a net negative charge, other biological molecules applied to the surface are prone to stick by non-specific electrostatic attraction. For example, DNA "probes" which are single strands are often contacted with an array of DNA single strands affixed to a solid support. Since the probe has a known nucleotide sequence and since a particular single strand of DNA will bind preferentially to a complementary strand, the particular immobilized strand to which the probe reacts reveals the nucleotide sequence of the previously unknown immobilized strand. Yet simple experiments of this type (probe studies) are severely confounded by electrostatic sticking of the probe to the derivatized (hence electrostatically charged) glass surface. For instance, the probe is often radiolabeled so that its presence can be detected by an ordinary radiation detector. Thus, the location of the probe on the glass surface, as evidenced by the detector, reveals the chemical identity or sequence of the immobilized DNA strand at that particular location on the glass surface (which is known and designated in advance). Yet the radiation detector is unable to distinguish between probe that is chemically bound to a complementary strand of DNA affixed to the solid support, and probe that is simply electrostatically stuck to the glass surface (but not to a DNA strand).

Derivatized surfaces can also result in what shall be known as "spreading." Spreading occurs because the solid support surface becomes hydrophilic upon derivatization. As a result, when a biological molecule, such as a DNA (desired to be immobilized upon the solid support) is contacted with the surface of the solid support, it spreads rather than remaining in a discrete "spot." Spreading is a major constraint on array density, i.e., the number of different "spots" or "clusters of biological molecules that can be arranged on a single solid support surface. Hence, any means to curtail spreading, and so increase array density, is highly desirable. SUMMARY The invention provides articles of manufacture comprising covalently bound molecules, e.g., biological molecules, such as nucleic acids, or small molecules, on a substrate surface derivatized with a hydrophobic monolayer. The invention provides articles of manufacture comprising an array of covalently bound molecules on a substrate surface derivatized with a hydrophobic monolayer made by a method comprising the following steps: (a) providing a molecule covalently modified by reaction with a compound having the formula: Rl — X — R2 , wherein Rl is a cyclic ether group or an amino group, R2 is an alkoxysilane group and X is a moiety chemically suitable for linking the cyclic ether group or the amino group to the alkoxysilane group; (b) providing a substrate surface, wherein the surface is reactive with a silane group; (c) providing a hydrophobic composition, wherein the hydrophobic composition comprises a silanated end; (d) reacting a plurality of compositions of step (c) with the substrate surface of step (b) under conditions wherein the silanated end of the compositions become directly or indirectly covalently attached to the , substrate surface and the substrate surface is less than 100% saturated with compositions, thereby making a substrate surface derivatized with hydrophobic groups; and, (e) reacting the molecule of step (a) with the derivatized substrate surface such that the molecule is covalently attached to an underivatized portion of the substrate surface.

The invention provides an article of manufacture comprising covalently bound molecules on a substrate surface derivatized with a hydrophobic monolayer made by a method comprising the following steps: (a) providing a substrate surface, wherein the surface is reactive with a silane group, e.g., an alkyl silane group, an alkoxy silane group, a monohalosilane group, a dihalosilane group or a trihalosilane group; (b) providing a molecule capable of reacting with the substrate surface of step (a) such that the molecule becomes covalently bound to the substrate surface; (c) providing a composition comprising the general formula selected from the group consisting of an alkyl silane, an alkoxy silane, and a group having the general formula (X)₃-Si-R, H(X)₂-Si-R and or H₂X-Si-R, wherein R comprises an alkyl group and X is a halogen selected from the group consisting of CI, Br, I and F; (d) reacting a plurality of compositions of step (c) with the substrate surface of step (a) under conditions wherein the silanated end of the compositions become directly or indirectly covalently attached to the substrate surface and the substrate surface is less than 100% saturated with compositions, thereby making a substrate surface derivatized with hydrophobic groups; and, (e) reacting the molecule of step (b) with the derivatized substrate surface such that the molecule is covalently attached to an underivatized portion of the substrate surface. In one aspect, the molecule of step (b) is reacted with the derivatized substrate surface such that a plurality of substantially identical molecules are attached to the surface on at least one discrete and known location to form a cluster of substantially identical molecules. The plurality of clusters can be spotted onto the derivatized substrate surface to form an array of clusters. The molecule of step (b) can comprise a nucleic acid, a polypeptide, a lipid, a polysaccharide or a small molecule. In alternative aspects, the composition of step (c) comprises a bis-triethoxyl octyl silane or a mono-trichloro octyl silane.

The invention provides an article of manufacture comprising an array of covalently bound molecules on a substrate surface derivatized with a hydrophobic monolayer and is made by a method comprising the following steps: (a) providing a molecule covalently modified by reaction with a compound having the formula: Ri — X — R₂ , wherein Ri is a cyclic ether group or an amino group, R2 is an alkoxysilane group and X is a moiety chemically suitable for linking the cyclic ether group or the amino group to the alkoxysilane group; (b) providing a substrate surface, wherein the surface is reactive with a monohalosilane group, a dihalosilane group or a trihalosilane group; (c) providing a composition comprising the general formula selected from the group consisting of an alkyl silane, an alkoxy silane, and a group having the general formula (X)₃ -Si-R, H(X)₂-Si-R and/or H₂X-Si-R, wherein R comprises an alkyl group and X is a halogen selected from the group consisting of CI, Br, I and F; (d) reacting a plurality of compositions of step (c) with the substrate surface of step (b) under conditions wherein the silanated end of the compositions become directly or indirectly covalently attached to the substrate surface and the substrate surface is less than 100% saturated with compositions, thereby making a substrate surface derivatized with hydrophobic groups; and, (e) reacting the molecule of step (a) with the derivatized substrate surface such that the molecule is covalently attached to an underivatized portion of the substrate surface. In alternative aspects, the composition of step (c) comprises a bis-triethoxyl octyl silane or a mono-trichloro octyl silane.

The invention also provides articles of manufacture made by a method comprising the following steps: (a) providing a molecule, e.g., a modified biological molecule, comprising a molecule, e.g., a biological molecule, modified by reaction with a compound having the formula: Ri — X — R₂ , wherein Ri is a cyclic ether group or an amino group, R₂ is an alkoxysilane group and X is a moiety chemically suitable for linking the cyclic ether group or the amino group to the alkoxysilane group; (b) providing a substrate surface, wherein the surface is reactive with a monohalosilane group, a dihalosilane group or a trihalosilane group; (c) providing a composition comprising the general formula selected from the group consisting of an alkyl silane, an alkoxy silane, and a group having the general formula (X)₃ -Si-R, H(X)₂-Si-R and/or H₂X-Si-R, wherein R comprises an alkyl group and X is a halogen selected from the group consisting of CI, Br, I and F; (d) reacting a plurality of compositions of step (c) with the substrate surface of step (b) under conditions wherein the silanated end of the compositions become directly or indirectly covalently attached to the substrate surface and the substrate surface is less than 100% saturated with compositions, thereby making a substrate surface substantially derivatized with hydrophobic groups; and, (e) reacting the modified molecule of step (a) with the derivatized substrate surface. In one aspect, the reaction of step (d) generates a hydrophobic monolayer upon which the modified molecules of step (a) are deposited, or "spotted." In one aspect, the molecule of step (a) is reacted with the derivatized substrate surface such that the molecule is covalently attached to an underivatized portion of the substrate surface. In alternative aspects, the composition of step (c) comprises a bis-triethoxyl octyl silane or a mono- trichloro octyl silane. one aspect, the modified molecule, e.g., biological molecule, is reacted with the derivatized substrate surface such that a plurality of substantially identical molecules are attached to the surface on at least one discrete and known location to form a cluster (e.g., "spot" or "biosite") of substantially identical molecules. The plurality of clusters can be spotted, e.g., printed, onto the derivatized substrate surface. The plurality of spotted clusters or "biosites" can comprise an array of clusters of substantially identical molecules, e.g., biological molecules, small molecules, and the like.

In one aspect, the molecule of step (c) is reacted with the derivatized substrate surface under anhydrous conditions. The anhydrous conditions can comprise an anhydrous ethanol solution. The molecule of step (c) can be in an anhydrous solution, such as an anhydrous ethanol solution. The molecule of step (c) can be in a solution at an amount of about 0.0005% to about 0.002% v/v. The molecule of step (c) can be reacted with the derivatized substrate surface at about room temperature. The molecule of step (c) can be reacted with the derivatized substrate surface for about 24 to 48 hours. In one aspect, the substrate surface comprises a composition comprising a surface-exposed hydroxyl group. The substrate surface can comprise a silicon dioxide. The substrate surface can comprise a glass. In alternative aspects, the substrate surface comprises a mica, a quartz, an alumina, a titania, SN0₂, RU0₂, Pt0₂, or a metal oxide. In alternative aspects, the substrate surface is 99.9% derivatized with hydrophobic groups, 99.5% derivatized with hydrophobic groups, 99% derivatized with hydrophobic groups, 95% derivatized with hydrophobic groups, 90% derivatized with hydrophobic groups, 80% derivatized with hydrophobic groups, 70% derivatized with hydrophobic groups, and 60% derivatized with hydrophobic groups.

In alternative aspects, the composition of step (c) is n-octyltrichlorosilane or n- hexyltrichlorosilane. In step (c), the alkyl group can comprise the general formula C_nH_(2n+i), wherein n is an integer between 2 and about 30. Alternatively, in step (c), the alkyl group can comprise between about 3 and about 35 carbons. The alkyl group can also comprise an unbranched or a branched alkyl chain. The alkyl group can comprise an alkane group or an alkene group. The alkyl group can comprise an unsaturated or a saturated alkyl chain. The alkyl group can comprise a substituted alkyl group. hi alternative aspects, the alkyl group comprises between about 2 and about 40 carbons, between about 3 and about 30 carbons, between about 4 and about 20 carbons, between about 8 and about 15 carbons, and between about 10 and about 12 carbons. hi one aspect, the cyclic ether is a compound comprising an epoxide group, such as ethylene oxide. The cyclic ether can also be an oxirane group. The cyclic ether can be a compound comprising an aromatic hydrocarbon epoxide group. hi one aspect, the Ri group reacts with a molecule, e.g., a biological molecule, a small molecule and the like. The Ri group can be covalently bound to the molecule, e.g., biological molecule or small molecule.

In alternative aspects, the cyclic ether is an epoxide group and the alkoxysilane can be — Si(OCH₃)₃, — Si(OC₂ H₅)₃, — Si(OCH₃)H₂, — Si(OCH₃)(CH₃) ₂, or — Si(OCH₃)₂CH₃. The cyclic ether can be an epoxide group and the compound can be a 3- glycidoxypropyltrimethoxysilane (GPTS).

In one aspect, the Ri amino group comprises a primary amino group. The Ri can be an amino group and the alkoxysilane can be selected from the group consisting of — Si(OCH₃)₃, — Si(OC₂ H₅)₃ and

Ri

— Si— R ^■2 ,

R₃ wherein Ri, R₂ and R₃ are selected from the group consisting of — H, — CH₃, — OCH₃, and — OC₂ H₃, and provided that at least one of Ri, R or R₃ is either

— OCH₃ or — OC₂H₃. In one aspect, the Ri is an amino group and the compound is 3-aminopropyltriethoxysilane.

In alternative aspects, the molecule that is reacted with (e.g., "spotted onto") the derivatized substrate surface (e.g., the hydrophobic monolayered surface) is a biological molecule and a small molecule. The biological molecule can comprise a polypeptide, a peptide or a peptidomimetic. The biological molecule can comprise a polysaccharide, or an analog or a mimetic thereof. The biological molecule can comprise a lipid, or an analog or a mimetic thereof. The biological molecule can comprise a nucleic acid or an analog or mimetic thereof, such as a DNA, a cDNA, an oligonucleotide, a genomic fragment or an RNA, such as an mRNA.

In one aspect, when the biological molecule is a nucleic acid that is reacted with (e.g., "spotted onto") the derivatized substrate surface, the nucleic acid is reacted with the Ri group at its 5' end. The nucleic acid can also comprise a cloning vehicle, such as a bacterial artificial chromosome (BAG), aplasmid, a cosmid, a bacteriophage PI -derived vector (PAC), a yeast artificial chromosome (YAC), a mammalian artificial chromosome (MAC) or an engineered virus or phage. The details of one or more aspects of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

All publications, GenBank Accession references (sequences), ATCC Deposits, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes.

DESCRIPTION OF DRAWINGS Figure 1 graphically shows the results of measurements of the diameters spots on a substrate surface, where the spots comprise a nucleic acid immobilized onto a derivatized substrate surface using exemplary compositions and methods of the invention, as described in detail in Example 1, below.

Like reference symbols in the various drawings indicate like elements. DETAILED DESCRIPTION The invention provides articles of manufacture comprising covalently bound compositions, e.g., biological molecules, on substrate surfaces that have been derivatized with a hydrophobic monolayer. The articles of manufacture are made by a method comprising first derivatizing a substrate surface with hydrophobic groups followed by addition of derivatized compositions, e.g., biological molecules. Any hydrophobic group can be used to derivatize the substrate surfaces to form a hydrophobic monolayer. The hydrophobic groups used to derivatize a substrate surface can comprise alkyl silanes, alkoxy silanes and/or compositions comprising (X)₃-Si-R, H(X)₂-Si-R and or H X-Si-R, wherein R comprises an alkyl group and X is a halogen, e.g., CI, Br, I and/or F.

Pre-derivatization of the surface with hydrophobic groups helps control the final density and spot morphology of surface-immobilized molecules. This is particularly useful in "gene chip" (i.e., array or biochip) manufacturing and applications, where nucleic acids are surface-immobilized. Deposition of derivatized compositions, e.g., biological molecules using the methods of the invention increases the stability, uniformity, and consistency of the "clusters" or "spots" of surface-immobilized molecules. The methods of the invention are particularly useful for increasing the stability, uniformity, and consistency of "spots" of biological molecules placed onto a surface using contact liquid deposition. For example, making articles of manufacture, such as arrays, as described in U.S. Patent No. 6,048,695, further incorporating the methods of the instant invention results in an "array" or "chip" with increased stability, uniformity, and consistency of the "clusters" or "spots" of surface- immobilized biological molecules.

While the invention is not limited by any particular mechanism or theory of action, the increased the stability, uniformity, and consistency of the "clusters" or "spots" of surface-immobilized biological molecules may be because pre-derivatization of the surface with hydrophobic groups reduces the amount of surface energy. h one aspect of the invention, modified compositions, e.g., biological molecules, such as nucleic acids or polypeptides, are immobilized to a variety of solid surfaces that have been derivatized with a hydrophobic monolayer. The compositions or biological molecules can be derivatized with any composition. For example, in one aspect, the biological molecules used to make the articles of manufacture of the invention are derivatized with compounds having two crucial functionalities: a ring ether and an alkoxysilane group. The biological molecule (e.g., nucleic acid) is first reacted with the ring ether. Then the newly modified biological molecules (e.g., nucleic acids) are contacted with a surface (e.g., glass). The alkoxysilane groups (on the derivatized biological molecule) react with a hydroxyl-containing (e.g., hydroxyl derivatized) surface, e.g., Si~OH groups. These groups can be on a glass surface. In another aspect, the chemically modified biological molecules (e.g., nucleic acids) are derivatized with compounds having two crucial functionalities: an amino group and an alkoxysilane group. The biological molecules (e.g., nucleic acid) react with the amino group. Then the newly modified biological molecules (e.g., nucleic acids) are contacted with a substrate surface, e.g., glass. The alkoxysilane group (on the derivatized biological molecule) reacts with a hydroxyl-containing (e.g., hydroxyl derivatized) surface, e.g., Si~OH groups on the glass surface.

Any type of composition can be immobilized to a substrate surface in the compositions and methods of the invention, including synthetic inorganic or organic compositions (e.g. "small molecules" or molecules from combinatorial chemical libraries), compositions isolated or derived from natural sources, e.g., biological molecules, such as nucleic acids. Nucleic acids, e.g., DNA or RNA, are one of many types of biological compositions, e.g., biological polymers that can be used in the compositions and methods of the invention. A polymer can be a molecule that has joined prefabricated units, e.g., monomers or compositions that can be of limited diversity, linked together, usually by identical mechanisms, e.g., a cellulose is a polymer is simple sugars or polysaccharides.

Exemplary biological molecules include but are not limited to DNA, RNA, protein, peptides, lipids, saccharides, polysaccharides and mimetics and analogs thereof. Thus, a skilled artisan recognizes that any biological molecule, including those having a structure found in nature or a synthetic structure, including polymers, can be modified and used in the compositions and methods of the invention to be affixed to a solid surface.

Any substrate surface that can react with an alkoxysilane and or a monohalo-, a dihalo- or a trihalosilane group can be used in the compositions and methods of the invention. For example, exemplary substrate surfaces that can be used in the compositions and methods of the invention comprise reactive functional groups, e.g., hydroxyl groups. These include, though are not limited to: quartz glass, mica, alumina (Al₂O₃, titania (TiO₂), SnO₂, RuO₂, PtO₂, plastics such as the following polymer materials, polystyrene, polyester, polycarbonate, polyethylene, polypropylene, and nylon as well as numerous semi-conductive surfaces, such as numerous other metal oxide surfaces and equivalents. Definitions

As used herein, the term "alkyl" is used to refer to a branched or unbranched, saturated or unsaturated, univalent or bivalent hydrocarbon radical. In alternative aspect, the alkyl an have from 1 to about 30 carbons, or, from about 4 to about 20 carbons, or, from about 6 to about 18 carbons. When the alkyl group has from 1 to about 6 carbon atoms, it can be referred to as a "lower alkyl." Suitable alkyl radicals include, for example, structures containing one or more methylene, methine and/or methyne groups. The term also includes branched structures have a branching motif similar to i-propyl, t-butyl, i-butyl, 2-ethylpropyl, etc. As used herein, the term encompasses "substituted alkyls." "Substituted alkyl" refers to an alkyl as just described including one or more functional groups such as lower alkyl, aryl, acyl, halogen (i.e., alkylhalos), hydroxy, amino, alkoxy, alkylamino, acylamino, thioamido, acyloxy, aryloxy, aryloxyalkyl, mercapto, thia, aza, oxo, both saturated and unsaturated cyclic hydrocarbons, heterocycles and the like. These groups maybe attached to any carbon of the alkyl moiety. Additionally, these groups may be pendent from, or integral to, the alkyl chain. As used herein, the term "arene" refers to any substituted or unsubstituted mono- or polycyclic aromatic hydrocarbon compound as well as any mono- or polycyclic heteroaromatic compounds, and can include fused or bridged ring systems.

The term "nucleic acid" as used herein refers to a deoxyribonucleotide (DNA) or ribonucleotide (RNA) in either single- or double-stranded form. The term encompasses nucleic acids containing known analogues of natural nucleotides. The term encompasses mixed oligonucleotides comprising an RNA portion bearing 2'-O-alkyl substituents conjugated to a DNA portion via a phosphodiester linkage, see, e.g., U.S. Patent No. 5,013,830. The term also encompasses nucleic-acid-like structures with synthetic backbones. DNA backbone analogues provided by the invention include phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioacetal, methylene(methylimino), 3'-N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs); see Oligonucleotides and Analogues, a Practical Approach, edited by F. Eckstein, IRL Press at Oxford University Press (1991); Antisense Strategies, Annals of the New York Academy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan (1993) J. Med. Chem. 36:1923-1937;

Antisense Research and Applications (1993, CRC Press). PNAs contain non-ionic backbones, such as N-(2-aminoethyl) glycine units. Phosphorothioate linkages are described, e.g., by U.S. Patent Nos. 6,031,092; 6,001,982; 5,684,148; see also, WO 97/03211; WO

96/39154; Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197. Other synthetic backbones encompassed by the term include methyl-phosphonate linkages or alternating methylphosphonate and phosphodiester linkages (see, e.g., U.S. Patent No. 5,962,674; Strauss-Soukup (1997) Biochemistry 36:8692-8698), and benzylphosphonate linkages (see, e.g., U.S. Patent No. 5,532,226; Samstag (1996) Antisense Nucleic Acid Drug Dev 6:153- 156). The term nucleic acid is used interchangeably with gene, DNA, RNA, cDNA, mRNA, oligonucleotide primer, probe and amplification product.

The terms "polypeptide," "protein," and "peptide" include compositions of the invention that also include "analogs," or "conservative variants" and "mimetics" or "peptidomimetics" with structures and activity that substantially correspond to the polypeptide from which the variant was derived, as discussed in detail, below.

The term "small molecule" means any synthetic small molecule, such as an organic molecule or a synthetic molecule, such as those generated by combinatorial chemistry methodologies. These small molecules can be synthesized using a variety of procedures and methodologies, which are well described in the scientific and patent literature, e.g., Organic Syntheses Collective Nolumes, Gilman et al. (Eds) John Wiley & Sons, Inc., ΝY; Nenuti (1989) Pharm Res. 6:867-873. Synthesis of small molecules, as with all other procedures associated with this invention, can be practiced in conjunction with any method or protocol known in the art. For example, preparation and screening of combinatorial chemical libraries are well known, see, e.g., U.S. Patent Νos. 6,096,496; 6,075,166; 6,054,047; 6,004,617; 5,985,356; 5,980,839; 5,917,185; 5,767,238.

The terms "array" or "microarray" or "biochip" or "chip" as used herein is an article of manufacture, a device, comprising a plurality of immobilized target elements, each target element comprising a "cluster" or "spot" or defined area comprising a particular composition, such as a biological molecule (e.g., a nucleic acid molecule or polypeptide, such as an antibody) or a small molecule immobilized to a solid surface, as discussed in further detail, below.

Generating and Manipulating Nucleic Acids

The invention provides articles of manufacture comprising arrays that include modified nucleic acid compositions and methods for making and using these arrays. The nucleic acid can be modified by reaction with a compound having the formula: Ri — X — R₂ , where Ri is a cyclic ether group or an amino group, R₂ is an alkoxysilane group and X is a moiety chemically suitable for linking the cyclic ether group or the amino group to the alkoxysilane group. The modified nucleic acid or the immobilized nucleic acid on the array can be representative of genomic DNA, including defined parts of, or entire, chromosomes, or entire genomes. In several aspects, the arrays and methods of the invention are used in comparative genomic hybridization (CGH) reactions, including CGH reactions on arrays (see, e.g., U.S. Patent Nos. 5,830,645; 5,976,790). The invention can be practiced in conjunction with any method or protocol or device known in the art, which are well described in the scientific and patent literature.

General Techniques

The biological molecules, including the nucleic acids, used to practice this invention, whether RNA, cDNA, genomic DNA, vectors, viruses or hybrids thereof, may be isolated from a variety of sources, genetically engineered, amplified, and or expressed/ generated recombinantly (recombinant polypeptides can be modified or immobilized to arrays in accordance with the invention). Any recombinant expression system can be used, including bacterial, mammalian, yeast, insect or plant cell expression systems.

Alternatively, these nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Carruthers (1982) Cold Spring Harbor Symp. Quant. Biol. 47:411-418; Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Terra. Lett. 22:1859; U.S. Patent No. 4,458,066. Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with a primer sequence.

Techniques for the manipulation of nucleic acids, such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, hie, New York (1997);

LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid Preparation, Tij ssen, ed. Elsevier, N.Y. (1993).

Another useful means of obtaining and manipulating nucleic acids used in the compositions and methods of the invention is to clone from genomic samples, and, if necessary, screen and re-clone inserts isolated (or amplified) from, e.g., genomic clones or cDNA clones or other sources of complete genomic DNA. Sources of nucleic acid used in the methods and compositions of the invention (e.g., nucleic acid spotted onto a hydrophobic- derivatized substrate surface) include genomic or cDNA libraries contained in, or comprised entirely of, e.g., mammalian artificial chromosomes (see, e.g., Ascenzioni (1997) Cancer Lett. 118:135-142; U.S. Patent Nos. 5,721,118; 6,025,155) (including human artificial chromosomes, see, e.g., Warburton (1997) Nature 386:553-555; Roush (1997) Science 276:38-39; Rosenfeld (1997) Nat. Genet. 15:333-335); yeast artificial chromosomes (YAC); bacterial artificial chromosomes (BAG); PI artificial chromosomes (see, e.g., Woon (1998) Genomics 50:306-316; Boren (1996) Genome Res. 6:1123-1130); PACs (a bacteriophage Pl- derived vector, see, e.g., Ioannou (1994) Nature Genet. 6:84-89; Reid (1997) Genomics 43:366-375; Nothwang (1997) Genomics 41:370-378; Kern (1997) Biotechniques 23:120- 124); cosmids, plasmids or cDNAs.

The compositions, e.g., biological molecules, such as nucleic acids, can be deposited as "spots" or "clusters" or "biosites" on substrate surfaces using any protocol, see, e.g., the U.S. Patents cited herein.

Amplification of Nucleic Acids

Amplification using oligonucleotide primers can be used to generate nucleic acids used in the compositions and methods of the invention, to detect or measure levels of test or control samples hybridized to an array, and the like. The skilled artisan can select and design suitable oligonucleotide amplification primers. Amplification methods are also well known in the art, and include, e.g., polymerase chain reaction, PCR (PCR PROTOCOLS, A

GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N.Y. (1990) and

PCR STRATEGIES (1995), ed. Innis, Academic Press, Inc., N.Y., ligase chain reaction (LCR) (see, e.g., Wu (1989) Genomics 4:560; Landegren (1988) Science 241 : 1077; Barringer

(1990) Gene 89:117); transcription amplification (see, e.g., Kwoh (1989) Proc. Natl. Acad.

Sci. USA 86:1173); and, self-sustained sequence replication (see, e.g., Guatelli (1990) Proc.

Natl. Acad. Sci. USA 87:1874); Q Beta replicase amplification (see, e.g., Smith (1997) J.

Clin. Microbiol. 35:1477-1491), automated Q-beta replicase amplification assay (see, e.g., Burg (1996) Mol. Cell. Probes 10:257-271) and other RNA polymerase mediated techniques

(e.g., NASBA, Cangene, Mississauga, Ontario); see also Berger (1987) Methods Enzymol.

152:307-316; Sambrook; Ausubel; U.S. Patent Nos. 4,683,195 and 4,683,202; Sooknanan

(1995) Biotechnology 13:563-564. Polypeptides

The invention provides articles of manufacture comprising arrays with immobilized polypeptides, peptides and peptidomimetics. The polypeptides can be modified by reaction with a compound having the formula: Ri — X — R₂ , where Ri is a cyclic ether group or an amino group, R₂ is an alkoxysilane group and X is a moiety chemically suitable for linking the cyclic ether group or the amino group to the alkoxysilane group. As noted above, the terms "polypeptide," "protein," and "peptide," used to practice the invention, include compositions of the invention that also include "analogs," or "conservative variants" and "mimetics" or "peptidomimetics." The terms "mimetic" and "peptidomimetic" refer to a synthetic chemical compounds. The mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids. The mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetics' structure and/or activity. Polypeptide mimetic compositions can contain any combination of non-natural structural components, which are typically from three structural groups: a) residue linkage groups other than the natural amide bond ("peptide bond") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like. A polypeptide can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds. Individual peptidomimetic residues can be joined by peptide bonds, other chemical bonds or coupling means, such as, e.g., glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N,N'-dicyclohexylcarbodiimide (DCC) or N,N'-diisopropylcarbodiimide (DIC). Linking groups that can be an alternative to the traditional amide bond ("peptide bond") linkages include, e.g., ketomethylene (e.g., -C(=O)-CH₂- for -C(=O)-NH-), aminomethylene (CH₂-NH), ethylene, olefin (CH=CH), ether (CH₂-O), thioether (CH₂-S), tetrazole (CN₄-), thiazole, retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357, "Peptide Backbone Modifications," Marcell Dekker, NY). A polypeptide can also be characterized as a mimetic by containing all or some non-natural residues in place of naturally occurring amino acid residues; non-natural residues are well described in the scientific and patent literature. The skilled artisan will recognize that individual synthetic residues and polypeptides incorporating mimetics can be synthesized using a variety of procedures and methodologies, which are well described in the scientific and patent literature, e.g., Organic Syntheses Collective Nolumes, Gilman, et al., Organic Syntheses Collective Nolumes, Gilman et al. (Eds) John Wiley & Sons, Inc., ΝY. Polypeptides incorporating mimetics can also be made using solid phase synthetic procedures, as described, e.g., by U.S. Pat. No. 5,422,426. Peptides and peptide mimetics can also be synthesized using combinatorial methodologies. Various techniques for generation of peptide and peptidomimetic libraries are well known, and include, e.g., multipin, tea bag, and split-couple-mix techniques; see, e.g., al-Obeidi (1998) Mol. Biotechnol. 9:205-223; Hruby (1997) Curr. Opin. Chem. Biol. 1:114-119; Ostergaard (1997) Mol. Divers. 3:17-27; Ostresh (1996) Methods Enzymol. 267:220-234. Modified polypeptide and peptides can be further produced by chemical modification methods, see, e.g., Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896. These peptides can also be synthesized, whole or in part, using chemical methods well known in the art (see e.g., Caruthers (1980) Nucleic Acids Res. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser. 225-232; Banga, A.K., Therapeutic Peptides and Proteins,

Formulation, Processing and Delivery Systems (1995) Technomic Publishing Co., Lancaster, PA. Peptide synthesis can be performed using various solid-phase techniques (see e.g., Roberge (1995) Science 269:202; Merrifield (1997) Methods Enzymol. 289:3-13) and automated synthesis may be used. The compositions, e.g., biological molecules, such as polypeptides, can be deposited as "spots" or "clusters" on substrate surfaces using any protocol, see, e.g., the U.S. Patents cited herein.

Arrays, or "BioChips"

The invention provides articles of manufacture comprising covalently bound compositions, e.g., biological molecules, on substrate surfaces that have been derivatized with a hydrophobic monolayer. For example, the articles of manufacture can be "arrays" or "microarrays" or "biochips" or "chip" comprising modified biological molecules, such as modified nucleic acids or polypeptides.

Arrays are generically a plurality of target elements immobilized onto the surface of the array as defined "spots," "clusters," or "biosites," each target element comprising a one or more biological molecules (e.g., nucleic acids or polypeptides) immobilized a solid surface for association (e.g., specific binding or hybridization) to a sample. The immobilized nucleic acids can contain sequences from specific messages (e.g., as cDNA libraries) or genes (e.g., genomic libraries), including a human genome. Other target elements can contain reference sequences and the like. The biological molecules of the arrays may be arranged on the solid surface at different sizes and different densities. The densities of the biological molecules in a cluster and the number of clusters on the array will depend upon a number of factors, such as the nature of the label, the solid support, the degree of hydrophobicity of the substrate surface, and the like. Each cluster/ biosite may comprise substantially the same biological molecule (e.g., nucleic acid or polypeptide), or, a mixture of biological molecules (e.g., nucleic acids of different lengths and/or sequences). Thus, for example, a cluster/ biosite may contain more than one copy of a cloned piece of DNA, and each copy may be broken into fragments of different lengths.

The surface onto which the modified biological molecules of the invention are immobilized can include nitrocellulose, glass, quartz, fused silica, plastics and the like, as discussed further, below. The compositions and methods of the invention can incorporate in whole or in part designs of arrays, and associated components and methods of making and using arrays, as described, e.g., in U.S. Patent Nos. 6,197,503; 6,174,684; 6,159,685;

6,156,501; 6,093,370; 6,087,112; 6,087,103; 6,087,102; 6,083,697; 6,080,585; 6,054,270; 6,048,695; 6,045,996; 6,022,963; 6,013,440; 5,959,098; 5,856,174; 5,843,655; 5,837,832; 5,830,645; 5,770,456; 5,723,320; 5,700,637; 5,695,940; 5,556,752; 5,143,854; see also, e.g., WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; WO 89/10977; see also, e.g., Johnston (1998) Curr. Biol. 8:R171-R174; Schummer (1997) Biotechniques 23:1087-1092; Kern (1997) Biotechniques 23:120-124; Solinas-Toldo (1997) Genes, Chromosomes & Cancer 20:399-407; Bowtell (1999) Nature Genetics Supp. 21:25-32; Epstein (2000) Current Opinion in Biotech. 11:36-41; Mendoza (1999 Biotechniques 27: 778-788; Lueking (1999) Anal. Biochem. 270:103-111; Davies (1999) Biotechniques 27:1258-1261. Substrate Surfaces

Any substrate surface that can react with an alkoxysilane and/or a monohalo-, a dihalo- or a trihalosilane group can be used in the compositions and methods of the invention, including, e.g., glass (see, e.g., U.S. Patent No. 5,843,767), ceramics, quartz. The articles of manufacture of the invention can have substrate surfaces of a rigid, semi-rigid or flexible material. The substrate surface can be flat or planar, be shaped as wells, raised regions, etched trenches, pores, beads, filaments, or the like. Substrate surfaces can also comprise various materials such as paper, crystalline substrates (e.g. gallium arsenide), metals, metalloids, polacryloylmorpholide, various plastics and plastic copolymers, Nylon™, Teflon™, polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polystyrene/ latex, polymethacrylate, poly(ethylene terephthalate), rayon, nylon, poly(vinyl butyrate); polyvinylidene difluoride (PNDF) (see, e.g., U.S. Patent No. 6,024,872), silicones (see, e.g., U.S. Patent No. 6,096,817), polyformaldehyde (see, e.g., U.S. Patent Nos. 4,355,153; 4,652,613), cellulose (see, e.g., U.S. Patent No. 5,068,269), cellulose acetate (see, e.g., U.S. Patent No. 6,048,457), nitrocellulose, various membranes and gels (e.g., silica aerogels, see, e.g., U.S. Patent No. 5,795,557), paramagnetic or superparamagnetic microparticles (see, e.g., U.S. Patent No. 5,939,261) and the like. Silane (e.g., mono- and dihydroxyalkylsilanes, aminoalkyltrialkoxysilanes, 3-aminopropyl-triethoxysilane, 3-aminopropyltrimethoxysilane) can provide a hydroxyl functional group for reaction with an amine functional group.

The articles of manufacture comprise covalently bound molecules on substrate surfaces that have been derivatized with a hydrophobic monolayer. The hydrophobic monolayer is made by depositing on the substrate surface a composition comprising the general formula selected from the group consisting of (X)₃-Si-R, H(X)₂-Si-R and H₂X-Si-R, wherein R comprises an alkyl group and X is a halogen selected from the group consisting of CI, Br, I and F. These a composition can be made using any protocol, see, e.g., Gilman, Organic Syntheses Collective Volumes, Gilman et al. (Eds) John Wiley & Sons, Inc., NY. The hydrophobic monolayer (or "hydrophobic film") can be made using any protocol, see, e.g., the U.S. Patents cited herein.

EXAMPLES The following example is offered to illustrate, but not to limit the claimed invention. Example 1: Preparation and Using an Exemplary Array of the Invention The following example describes making and using an exemplary article of manufacture of the invention.

Preparing a hydrophobic monolayer

A solid surface, such as glass, is cleaned and coated with an n- octyltrichlorosilane solution. A bis-triethoxyl octyl silane solution can also be used. First, n- octyltrichlorosilane (or bis-triethoxyl octyl silane) is diluted in n-octyltrichlorosilane to 1% V/V in 100% ethanol. Next, the 1% n-octyltrichlorosilane solution is diluted to 0.0004% V/V in 1 L 100% ethanol pre- warmed at 65°C. Slides are put in a slide rack. All slides are covered with this 0.0004% n-octyltrichlorosilane/ethanol solution. Slides are incubated at 65 °C for about 14 hr. After the overnight treatment, the containers with the slides are cooled down to room temperature.

Two separate clean containers are filled with 95-100% room temperature ethanol. The treated slides are washed twice using this ethanol solution. Centrifuges are cleaned for plate spinning. The slides are spin dried for 5 minutes at 550 rpm. Slides are stored in a desiccator at room temperature.

This procedure forms a hydrophobic monolayer with fixed spacing of molecules. The tethered oligonucleotides were deposited with a fixed diameter of 200 microns. The hydrophobic monolayer in turn allows the modified biomolecules with silanized tethers to be attached directly onto the solid surface while the spot morphology and buffering spaces are controlled by hydrophobicity of the monolayer. Since this compound is mono-functional, the surface retains the optimal non-reactivity during probe attachments and target hybridizations, and minimizes washing stringency. Oligonucleotide Probes

Oligonucleotides ("oligos" as sense probes) of human p53, bcl-2, and b-actin of 50- & 70-mers were obtained from MWG (Germany) and Operon (Chicago, IL) respectively. The lyophilized oligo pellets were re-suspended in 0.1M sodium phosphate buffer pH 7.0 at 10, 20, and 30 mg/ml and resuspended for 16 hours. The resuspended oligos were then cross linked with glycid (tether) at a concentration of 40 mM final for 3 hours at 65°C. The cross linked oligos were cooled to room temperature and isopropanol precipitated for 16 hours at -20°C. The oligo pellets were resuspended in 0.1M sodium phosphate buffer pH 7.0 and transferred to a 384 well printing plate.

Printing Using the n-octyltrichlorosilane (or bis-triethoxyl octyl silane) coated slides, oligonucleotides, PCR products, plasmids, BAG clones conjugated with tethers (glycid ethoxysilane) were printed onto the prepared slides. Using a GENERATION III ARRAY SPOTTER™ (Amersham Bioscience), each cross linked oligo was spotted onto the octane silanized surface at the density of 76 replicate spots at a 150 mm diameter with 100 mm buffer between each spot.

BAC DNAs were cross-linked and printed under the same conditions as the cross-linked oligos. After printing, the slides were baked at 80°C for 5 hours and desalted using ethanol for 16 hours. Spots were post-processed by incubating at 65°C for 12 to 16 hours in 80%) ethanol, then spin-dried.

Hybridization The tethered oligonucleotides were tested over a 14-day post-octane treatment.

Target sequences were labeled with Cy3 or Cy5 nucleotides. Target sequences were generated as the anti-sense sequences of the sense probes while incorporating Cyanine3- dCTP during syntheses (Operon, IL). At a concentration of 10 ng/ml, each target was resuspended in IX final concentration of Array Spotting Buffer (Arrayit.com, CA) and hybridized for 3 hours at 65°C. The slides were washed in 2X SSC, 0.1% SDS at 30°C for 5 minutes, IX SSC, 0.1% SDS at 30°C for 5 minutes, 0.5X SSC at 30°C for 5 minutes, and 0.2X SSC at 22°C for 1 minute sequentially and spun dry for 5 minutes at 550 RPM. The slides were scanned at 100% laser power and appropriate PMT to give 1:1 Cy5 to Cy3 ratio at a 10 mm resolution using an AXON 4000B™ laser confocal scanner. The resulting scanned images were analyzed using the GENEPIX PRO 3.0™ software and the diameters and feature intensities were calculated accordingly.

From the GENEPIX PRO 3.0™ analysis files, the diameters of all 76 spots were averaged and the standard deviations were calculated using Microsoft EXCEL™. Figure 1 graphically shows the results of measurements of the diameters spots on the slide surface. Figure 1 shows a result of consistent spot diameters when n-octyltrichlorosilane is used to treat substrate surfaces and the tethered oligonucleotides are deposited with a fixed diameter of 200 microns over 14 day post octane treatment. The diameters of all "control" spots, i.e., spot placed on slide surfaces that had not been pre-treated to generate a hydrophobic monolayer, all had average spot diameters of over 200 microns. Without the surface treatment, the spots did not exhibit consistent diameter and morphology. All spots on slides with an n-octyltrichlorosilane monolayer had average spot diameters of under 200 microns.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. An article of manufacture comprising an array of covalently bound molecules on a substrate surface derivatized with a hydrophobic monolayer made by a method comprising the following steps: (a) providing a molecule covalently modified by reaction with a compound having the formula: R_\ — X — R₂ , wherein Ri is a cyclic ether group or an amino group, R₂ is an alkoxysilane group and X is a moiety chemically suitable for linking the cyclic ether group or the amino group to the alkoxysilane group;

(b) providing a substrate surface, wherein the surface is reactive with a monohalosilane group, a dihalosilane group or a trihalosilane group;

(c) providing a composition comprising the general formula selected from the group consisting of an alkyl silane, an alkoxy silane and a group having the general formula (X)₃ -Si-R, H(X)₂-Si-R or H₂X-Si-R, wherein R comprises an alkyl group and X is a halogen selected from the group consisting of CI, Br, I and F; (d) reacting a plurality of compositions of step (c) with the substrate surface of step (b) under conditions wherein the silanated end of the compositions become directly or indirectly covalently attached to the substrate surface and the substrate surface is less than 100% saturated with compositions, thereby making a substrate surface derivatized with hydrophobic groups; and, (e) reacting the molecule of step (a) with the derivatized substrate surface such that the molecule is covalently attached to an underivatized portion of the substrate surface.

2. An article of manufacture comprising an array of covalently bound biological molecules on a substrate surface derivatized with a hydrophobic monolayer made by a method comprising the following steps:

(a) providing a modified biological molecule comprising a biological molecule modified by reaction with a compound having the formula: Ri — X — R₂ , wherein Ri is a cyclic ether group or an amino group, R₂ is an alkoxysilane group and X is a moiety chemically suitable for linking the cyclic ether group or the amino group to the alkoxysilane group;

(b) providing a substrate surface, wherein the surface is reactive with a monohalosilane group, a dihalosilane group or a trihalosilane group; (c) providing a composition comprising the general formula selected from the group consisting of an alkyl silane, an alkoxy silane, and a group having the general formula (X)₃ -Si-R, H(X)₂-Si-R or H₂X-Si-R, wherein R comprises an alkyl group and X is a halogen selected from the group consisting of CI, Br, I and F;

5 (d) reacting a plurality of compositions of step (c) with the substrate surface of step (b) under conditions wherein the silanated end of the compositions become directly or indirectly covalently attached to the substrate surface and the substrate surface is less than 100% saturated with compositions, thereby making a substrate surface substantially derivatized with hydrophobic groups; and, o (e) reacting the modified biological molecule of step (a) with the derivatized substrate surface.

3. The article of manufacture of claim 1 , wherein the modified molecule of step (a) is reacted with the derivatized substrate surface such that a plurality of 5 substantially identical molecules are attached to the surface on at least one discrete and known location to form a cluster of substantially identical molecules.

4. The article of manufacture of claim 3 , wherein a plurality of clusters are spotted onto the derivatized substrate surface. 0

5. The article of manufacture of claim 4, wherein the plurality of spotted clusters comprise an array of clusters of substantially identical molecules.

6. The article of manufacture of claim 1, wherein the molecule of step (c) 5 is reacted with the substrate surface under anhydrous conditions.

7. The article of manufacture of claim 6, wherein the anhydrous conditions comprise an anhydrous ethanol solution.

0 8. The article of manufacture of claim 1, wherein the molecule of step (c) is in an anhydrous solution.

9. The article of manufacture of claim 8, wherein the anhydrous solution comprises an anhydrous ethanol solution.

10. The article of manufacture of claim 1 , wherein the molecule of step (c) is in a solution at an amount of about 0.0005% to about 0.002% v/v.

11. The article of manufacture of claim 1 , wherein the molecule of step (c) is reacted with the substrate surface at about room temperature.

12. The article of manufacture of claim 11 , wherein the molecule of step (c) is reacted with the substrate surface for about 24 to 48 hours.

13. The article of manufacture of claim 1 , wherein the substrate surface comprises a composition comprising a surface-exposed hydroxyl group.

14. The article of manufacture of claim 13, wherein the substrate surface comprises a silcon dioxide.

15. The article of manufacture of claim 14, wherein the substrate surface comprises a glass.

16. The article of manufacture of claim 13, wherein the substrate surface comprises a mica, a quartz, an alumina, a titania, SN0 , RU0 , Pt0₂, or a metal oxide.

17. The article of manufacture of claim 1, wherein the substrate surface is 99.9% derivatized with hydrophobic groups.

18. The article of manufacture of claim 17, wherein the substrate surface is 99.5% derivatized with hydrophobic groups.

19. The article of manufacture of claim 18, wherein the substrate surface is 99% derivatized with hydrophobic groups.

20. The article of manufacture of claim 19, wherein the substrate surface is 95% derivatized with hydrophobic groups.

21. The article of manufacture of claim 20, wherein the substrate surface is 90% derivatized with hydrophobic groups.

22. The article of manufacture of claim 1 , wherein the composition of step (c) is n-octyltrichlorosilane or n-hexyltrichlorosilane.

23. The article of manufacture of claim 1, wherem in step (c) the alkyl group comprises the general formula C_nH(₂n₊!), wherein n is an integer between 2 and about 30.

24. The article of manufacture of claim 1, wherein the alkyl group comprises an unbranched alkyl chain or a branched alkyl chain.

25. The article of manufacture of claim 24, wherein in step (c) the alkyl group comprises between about 3 and about 35 carbons.

26. The article of manufacture of claim 25, wherein in step (c) the alkyl group comprises between about 4 and about 20 carbons.

27. The article of manufacture of claim 26, wherein in step (c) the alkyl group comprises between about 8 and about 15 carbons.

28. The article of manufacture of claim 1, wherein the alkyl group of the composition of step (c) comprises an alkane group or an alkene group.

29. The article of manufacture of claim 1, wherein the alkyl group of the composition of step (c) comprises a substituted alkyl group.

30. The article of manufacture of claim 1, wherein the cyclic ether is a compound comprising an epoxide group.

31. The article of manufacture of claim 30, wherein the epoxide is ethylene oxide.

32. The article of manufacture of claim 1, wherein the cyclic ether is an oxirane group.

33. The article of manufacture of claim 1, wherein the cyclic ether is a compound comprising an aromatic hydrocarbon epoxide group.

34. The article of manufacture of claim 1, wherein the Ri group reacts with the biological molecule.

35. The article of manufacture of claim 35, wherein the Ri group is covalently bound to the biological molecule.

36. The article of manufacture of claim 1, wherein cyclic ether is an epoxide group and the alkoxysilane is — Si(OCH₃)₃, — Si(OC₂ H₅)₃, — Si(OCH₃)H₂, — Si(OCH₃)(CH₃) ₂, or — Si(OCH₃)₂CH₃.

37. The article of manufacture of claim 1, wherein cyclic ether is an epoxide group and the compound is 3-glycidoxypropyltrimethoxysilane (GPTS).

38. The article of manufacture of claim 1, wherein the Ri amino group comprises a primary amino group.

39. The article of manufacture of claim 1, wherein Ri is an amino group and the alkoxysilane is selected from the group consisting of — Si(OCH₃)₃, — Si(OC₂ H₅)₃ and

Ri

Si — R ^•;2 ,

R₃

wherein Ri, R₂ and R₃ are selected from the group consisting of — H, — CH₃, — OCH₃, and — OC₂ H₃, and provided that at least one of R_l9 R₂ or R₃ is either

— OCH₃ or— OC₂H₃.

40. The article of manufacture of claim 1, wherein Ri is an amino group and the compound is 3-aminopropyltriethoxysilane.

41. The article of manufacture of claim 1 , wherein the biological molecule comprises a polypeptide, a peptide or a peptidomimetic.

42. The article of manufacture of claim 1, wherein the biological molecule comprises a polysaccharide, or an analog or a mimetic thereof.

43. The article of manufacture of claim 1, wherein the biological molecule comprises a lipid, or an analog or a mimetic thereof.

44. The article of manufacture of claim 1 , wherein the biological molecule comprises a small molecule.

45. The article of manufacture of claim 1 , wherein the biological molecule comprises a nucleic acid or an analog or mimetic thereof.

46. The article of manufacture of claim 45, wherein the nucleic acid comprises a DNA or an RNA.

47. The article of manufacture of claim 45 , wherein the nucleic acid reacts with the Ri group at its 5' end.

48. The article of manufacture of claim 45, wherein the nucleic acid is an oligonucleotide.

49. The article of manufacture of claim 45, wherein the nucleic acid comprises a cloning vehicle.

50. The article of manufacture of claim 49, wherein the cloning vehicle comprises a bacterial artificial chromosome (BAG).

51. The article of manufacture of claim 49, wherein the cloning vehicle comprises a plasmid, a cosmid, a bacteriophage PI -derived vector (PAC), a yeast artificial chromosome (YAC) or a mammalian artificial chromosome (MAC).

52. The article of manufacture of claim 1, wherein the composition of step

(c) comprises a bis-triethoxyl octyl silane or a mono-trichloro octyl silane.

53. An article of manufacture comprising covalently bound molecules on a substrate surface derivatized with a hydrophobic monolayer made by a method comprising the following steps:

(a) providing a substrate surface, wherein the surface is reactive with a monohalosilane group, a dihalosilane group or a trihalosilane group;

(b) providing a molecule capable of reacting with the substrate surface of step

(a) such that the molecule becomes covalently bound to the substrate surface; (c) providing a composition comprising the general formula selected from the group consisting of an alkyl silane, an alkoxy silane, and a group having the general formula (X)₃ -Si-R, H(X)₂-Si-R or H₂X-Si-R, wherein R comprises an alkyl group and X is a halogen selected from the group consisting of CI, Br, I and F;

(d) reacting a plurality of compositions of step (c) with the substrate surface of step (a) under conditions wherein the silanated end of the compositions become directly or indirectly covalently attached to the substrate surface and the substrate surface is less than 100% saturated with compositions, thereby making a substrate surface derivatized with hydrophobic groups; and,

(e) reacting the molecule of step (b) with the derivatized substrate surface such that the molecule is covalently attached to an underivatized portion of the substrate surface.

54. The article of manufacture of claim 53, wherein the molecule of step

(b) is reacted with the derivatized substrate surface such that a plurality of substantially identical molecules are attached to the surface on at least one discrete and known location to form a cluster of substantially identical molecules.

55. The article of manufacture of claim 54, wherein a plurality of clusters are spotted onto the derivatized substrate surface to form an array of clusters.

56. The article of manufacture of claim 53, wherein the molecule of step (b) comprises a nucleic acid, a polypeptide, a lipid, a polysaccharide or a small molecule.

57. The article of manufacture of claim 53, wherein the composition of step (c) comprises a bis-triethoxyl octyl silane or a mono-trichloro octyl silane.

58. A method for making an article of manufacture comprising the following steps:

(a) providing a molecule covalently modified by reaction with a compound having the formula: Ri — X — R₂ , wherein Ri is a cyclic ether group or an amino group, R₂ is an alkoxysilane group and X is a moiety chemically suitable for linking the cyclic ether group or the amino group to the alkoxysilane group; (b) providing a substrate surface, wherein the surface is reactive with a silane group;

(c) providing a hydrophobic composition, wherein the hydrophobic composition comprises a silanated end;

(d) reacting a plurality of compositions of step (c) with the substrate surface of step (b) under conditions wherein the silanated end of the compositions become directly or indirectly covalently attached to the substrate surface and the substrate surface is less than 100% saturated with compositions, thereby making a substrate surface derivatized with hydrophobic groups; and,

(e) reacting the molecule of step (a) with the derivatized substrate surface such that the molecule is covalently attached to an underivatized portion of the substrate surface.

59. A method for making an article of manufacture comprising the following steps: (a) providing a molecule covalently modified by reaction with a compound having the formula: Ri — X — R₂ , wherein Ri is a cyclic ether group or an amino group, R₂ is an alkoxysilane group and X is a moiety chemically suitable for linking the cyclic ether group or the amino group to the alkoxysilane group; (b) providing a substrate surface, wherein the surface is reactive with a monohalosilane group, a dihalosilane group or a trihalosilane group;

(c) providing a composition comprising the general formula selected from the group consisting of an alkyl silane, an alkoxy silane and a group having the general formula (X)₃ -Si-R, H(X)₂-Si-R or H₂X-Si-R, wherein R comprises an alkyl group and X is a halogen selected from the group consisting of CI, Br, I and F;

60. A method of making an article of manufacture comprising the following steps:

(a) providing a modified biological molecule comprising a biological molecule modified by reaction with a compound having the formula: K_\ — X — R₂ , wherein Ri is a cyclic ether group or an amino group, R₂ is an alkoxysilane group and X is a moiety chemically suitable for linking the cyclic ether group or the amino group to the alkoxysilane group;

(d) reacting a plurality of compositions of step (c) with the substrate surface of step (b) under conditions wherein the silanated end of the compositions become directly or indirectly covalently attached to the substrate surface and the substrate surface is less than 100% saturated with compositions, thereby making a substrate surface substantially derivatized with hydrophobic groups; and, (e) reacting the modified biological molecule of step (a) with the derivatized substrate surface.

61. The method of claim 58, claim 59 or claim 60, wherein the

5 composition of step (c) comprises a bis-triethoxyl octyl silane or a mono-trichloro octyl silane.

62. An article of manufacture comprising an array of covalently bound molecules on a substrate surface derivatized with a hydrophobic monolayer made by a method comprising the following steps: o (a) providing a molecule covalently modified by reaction with a compound having the formula: Ri — X — R₂ , wherein Ri is a cyclic ether group or an amino group, R₂ is an alkoxysilane group and X is a moiety chemically suitable for linking the cyclic ether group or the amino group to the alkoxysilane group;

(b) providing a substrate surface, wherein the surface is reactive with a silane 5 group;

(d) reacting a plurality of compositions of step (c) with the substrate surface of step (b) under conditions wherein the silanated end of the compositions become directly or 0 indirectly covalently attached to the substrate surface and the substrate surface is less than 100% saturated with compositions, thereby making a substrate surface derivatized with hydrophobic groups; and,

(e) reacting the molecule of step (a) with the derivatized substrate surface such that the molecule is covalently attached to an underivatized portion of the substrate 5 surface.

63. An article of manufacture comprising covalently bound molecules on a substrate surface derivatized with a hydrophobic monolayer made by a method comprising the following steps: 0 (a) providing a substrate surface, wherein the surface is reactive with a silane group;

(b) providing a molecule capable of reacting with the substrate surface of step

(a) such that the molecule becomes covalently bound to the substrate surface; (c) providing composition comprising a hydrophobic molecule capable of reacting with the substrate surface of step (a) such that the molecule becomes covalently bound to the substrate surface;