WO2008060268A2

WO2008060268A2 - Tailorable hydrophilic surface modification

Info

Publication number: WO2008060268A2
Application number: PCT/US2006/041805
Authority: WO
Inventors: Chad A. Mirkin; Robert Elghanian
Original assignee: Northwestern University
Priority date: 2005-10-28
Filing date: 2006-10-27
Publication date: 2008-05-22
Also published as: WO2008060268A3

Abstract

The present invention is directed to modified surfaces and methods of making modified surfaces. More particularly, the present invention is directed to hydrophobic surfaces modified with hydrophilic polymers and, optionally, target molecules. These modified surfaces can be used in detection assays or as a means to improve solubility of the unmodified surface.

Description

TAILORABLE HYDROPHILIC SURFACE MODIFICATION CROSS-REFERENCE TO RELATED APPLICATION

[0001 J This application claims the benefit of U.S. provisional application Nos. 60/731 ,129, filed October 28, 2005, and 60/733,105, filed November 3, 2005, each of which is incorporated by reference in its entirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

[0002] This invention was made with government support from the Air Force Office of Scientific Research, grant no. F49620-01- 1-0401. The government has certain rights in this invention.

BACKGROUND OFTHE DISCLOSURE

Field of the Disclosure

[0003] The present disclosure is directed to tailorable hydrophilic surfaces. More specifically, the present disclosure provides methods of producing surfaces, in situ, having activated elements for use in screening or detection assays.

Related Technology

[0004] Detection of analytes or molecules that interact with a target of interest has long been a basis for biomedical research and drug development. New technologies and research methods have been developed in response to a need for effectively and efficiently detecting these analytes and/or interactions. Immobilization of analytes for use in various assay systems is one such important area of development.

[0005] Attachment of target compounds or other molecules of interest to myriad surfaces has been achieved through a variety of methods, many of which involve numerous intermediates and long and inefficient syntheses. For example, the methods described in U.S. Patent No. 6,465,178 require the synthesis of various polymer intermediates having an appropriate photoreactive or thermochemically reactive functionality prior to attaching the polymer intermediates to an appropriate inert surface. Numerous synthetic intermediates and tedious preparations are required to practice the methods disclosed therein.

[0006] To date, methods of producing surfaces modified with hydrophilic polymers, suitable for detection assays and which do not involve synthetic intermediates have not been addressed. A need for providing such modified surfaces using commercially available reagents, which can be tailored to exhibit differing levels of reactive sites or reactive properties adaptable to a particular desired end, still exists in the art. SUMMARY

[0007] Disclosed herein are methods of modifying a surface, and modified surfaces prepared from the disclosed methods.

[0008] Thus, the present invention provides a method of modifying a surface comprising contacting (1) an unmodified surface with (2) a compound having an amino functional group and a second functional group capable of covalently bonding to said unmodified surface to form an aminated surface. An aminated surface is a surface modified with a compound having a terminal amino group, wherein that amino group is available for further reaction (e.g., is not a tertiary amine). The aminated surface then is contacted with a hydrophilic polymer, an optional coupling reagent, and.a crosslinking reagent to form the modified surface. Nonlimiting examples of the compound having an amino functional group include silyl alkylamines, thiol alkylamines, disulfide alkylamines, and any amine having a second functionality suitable for bonding to the unmodified surface to provide an aminated surface. In some embodiments, the unmodified surface is hydrophobic, e.g., a hydrophobic polymer.

[0009] The hydrophilic polymer can comprise at least two, and typically a plurality of, carboxylic acid or carboxylic acid ester functional groups. Typically, the hydrophilic polymer comprises polyacrylic acid, polycarboxylate, polysulfonate, polyphosphate, polyphosphonate, polyethylene glycol, polyvinyl alcohol, or mixtures thereof. The hydrophilic polymer typically has the carboxylic acid or ester functional groups pendant to the backbone of the polymer. The molecular weight of the hydrophilic polymer can be about 10 kDa to about 500 kDa. Preferably, the molecular weight of the hydrophilic polymer is less than 250 kDa, or less than 200 kDa.

[0010] The cross-linking agent typically is a diamine, but alternatively can be any compound having at least two reactive functional groups, e.g., any mixture of -SH, -S-S-, - OH, and -NH₂, such as dithiols, disulfides, diols, aminoalcohols, and the like. The two reactive functional groups can be separated by up to 20 carbon atoms, but alternatively, can be vicinal or geminal. Typical cross-linking agents are diamines, such as hydrazine or linear or branched C_2-15 diamines.

[0011] The unmodified surface can be any of a variety of surfaces in need of modification for a desired end use. Surfaces such as glass (e.g., glass slides or glass beads), hydrophobic polymeric beads or surfaces, or gold micro- or nanoparticles all are contemplated as surfaces in the disclosed methods. Hydrophobic polymeric beads or surfaces can be polystyrene, polyethylene, polybutylene, polypropylene, polymerized mixed olefins, polyterpene, polyisoprene, polyvinyltoluene, poly(α-methylstyrene), poly(o-methylstyrene), poly(m- methylstyrene), poly(p-methylstyrene), poly(dimethylphenylene oxide), polyurethane, polyvinyl chloride, or mixtures thereof.

[00121 In some embodiments, the modified surface is further contacted with an activating agent, which provides a modified surface capable of reacting with a target molecule. A variety of target molecules are contemplated for use in the disclosed methods, including proteins or polypeptides, nucleic acids, and small molecules. The resulting surfaces can be useful in screening or detecting assays. Typically, the activating agent is N-hydroxy succinimide, but can be any agent which activates a carboxylic acid.

[0013] In another aspect, a method of modifying a surface is provided, comprising the steps of contacting (1) an unmodified surface with (2) a compound having a carboxylic acid, ester, or anhydride functional group and a second functional group capable of covalently bonding to said unmodified surface to form a carboxylated surface. A carboxylated surface is a surface modified with a compound having a carboxylic acid, ester, or anhydride group, wherein that carboxylic acid, ester, or anhydride group is available for further reaction. The carboxylated surface then is contacted with a hydrophilic polymer, an optional coupling reagent, and a cross-linking reagent to form the modified surface, typically having a -CO₂H or -CO₂Ci-₃alkyl functional group. Nonlimiting examples of the compound having a carboxylic acid, ester, or anhydride group include silyl alkyl carboxylic acids or esters, thiol alkyl carboxylic acids or esters, and any carboxylic acids or ester having a second functionality suitable for bonding to the unmodified surface to provide a carboxylated surface. Anhydrides can also be used. Cross-linking agents are typically dicarboxylic acids, dicarboxylic esters, and/or anhydrides and include, but are not limited to, HO₂C-C_0- ₎₅alkylCO₂H, C_1-3alkylO₂C-Co-i₅alkylCO₂C_1-3alkyl, maleic anhydride, succinic anhydride, phthalic anhydride, or mixtures thereof. Cross-linking agents can optionally be substituted with subsituents such as, but not limited to, halo, hydroxy, alkoxy, cyano, amino, and the like. Any hydrophilic polymers having at least two, and typically a plurality of, functional groups capable of covalently bonding with a carboxylated surface can be used. Polyamines, polyalcohols (such as polyvinyl alcohols), or polymers having pendant amine, alcohol, and/or thiol functional groups are contemplated.

[0014] In embodiments where the desired end use is for a screening or detection assay, the method can be performed in a manner so as to provide isolated modified sites on the surface, which can be desired for modification of surfaces with numerous target molecules. For example, when a glass or plastic slide is to be used in a detection assay, multiple sites of modification can be prepared wherein each site is designed to interact with or covalently bind to a different target molecule. Isolation of the modified sites from each other is desirable so as to avoid overlap or "bleeding" of detection signals of adjacent sites on the glass surface in the detection assay.

[0015] In embodiments where the surface initially is carboxylated (i.e., modified with a carboxylic acid functionality such as an acid, ester, or anhydride), the hydrophilic polymer will have at least two, but typically a plurality of, amine, alcohol, and/or thiol functional groups, and the cross-linking agent will have at least two carboxylic acid functional groups. The choice of initial modification dictates the choice of functional groups on the hydrophilic polymer and the cross-linking agent. Similarly, the activating agent will be an agent capable of activating the functional group on the hydrophilic polymer. In embodiments where the functional group is an amine, alcohol, or thiol, the activating agent will be an agent suitable to increase reactivity of the amine, alcohol, or thiol for reaction with the target molecule. The target molecule will have a functional group which can react with an amine, alcohol, or thiol, such as a phosphate, carboxylic acid, carboxylic ester, and the like.

BRIEF DESCRIPTION OF THE FIGURES

[00161 FIG 1 shows a schematic of a method of producing modified surfaces, wherein the reagents are mixed together in one step to produce a random polymer covalently attached to a surface, wherein the polymer has reactive N-hydroxysuccinimide groups capable of reacting with an analyte;

[0017] FIG 2 shows a schematic of a method of using modified surfaces to attach target molecules such as a biomolecule (e.g., a nucleic acid);

[0018] FIG 3 shows a surface modified with various polymers that have not been cross- linked with cross-linking agent (e.g., C₀), and treated with various concentrations of target molecules;

[0019] FIG 4 shows a surface modified with various polymers that have been cross-linked with a C₈ cross- linking agent, then treated with various concentrations of target molecules;

[0020] FIG 5 shows a surface modified with various polymers that have been cross-linked with a Ci₂ cross-linking agent, then treated with various concentrations of target molecules; and [0021] FIG 6 shows images of two different surfaces imprinted with uncross-linked polymers (C₃) and polymers cross-linked with a C₈ cross-linking agent; each was then treated with a 1 nM solution of a target molecules.

[0022] While the disclosed methods are susceptible to embodiments in various forms, there are illustrated in the drawings (and will hereafter be described) specific embodiments, with the understanding that the disclosure is intended to be illustrative, and is not intended to limit the invention to the specific embodiments described and illustrated herein.

DETAILED DESCRIPTION

[0023] Disclosed herein are methods of preparing modified surfaces and use of these modified surfaces. In some embodiments, the modified surfaces are used in detection assays. Generally speaking, the disclosed methods modify surfaces in a one-step procedure from simple reagents. The surfaces are modified with hydrophilic polymers, hi certain embodiments, the hydrophilic polymers may be cross-linked with cross-linking reagents of varying alkyl lengths, which may affect the contact angle of the deposited hydrophilic polymer on the modified surface. In other embodiments, the surfaces may include embedded functionality such as electrodes, hi still other embodiments, the hydrophilic polymers may be modified with activating agents for attachment of target molecules.

[0024] Nonlimiting examples of suitable surfaces for use in the present methods include, but are not limited to, glass slides or other glass surfaces, polymer films, laminates, polymeric microparticles (including paramagnetic microparticles), metal and metal oxide surfaces, computer chips (such as silicon chips), natural and synthetic membranes, and other silica- based and plastic surfaces, hi some cases, wherein the surface is glass, the glass may be treated with compound having an amino functional group such as an amino alkylsilane or an amino alkylhalosilane, which provides a amino group capable of further reaction to the glass surface.

[0025] Other surfaces may be similarly modified to provide a such amines. For example, if the surface includes a gold micro- or nanoparticle surface, the surface may be modified with an amino alkylthiol to provide an amine functionality. Other functional groups may be introduced to the surface via similar approaches to provide, e.g., alcohols, carboxylic acids, thiols, sulfonic acids, and the like, to the surface for modification.

[0026] Animation, hydroxylation, carboxylation, etc. of a surface (such as a polymeric surface) can be accomplished using corona discharge or a plasma glow discharge. Such methods are disclosed in, for example, U.S. Patent Nos. 6,355,270; 6,140,127; and 6,053,171, each of which is incorporated in its entirety by reference herein.

[0027] Hydrophobic polymeric beads or surfaces can be, for example, polystyrene, polyethylene, polybutylene, polypropylene, polymerized mixed olefins, polyterpene, polyisoprene, polyvinyltoluene, poly(α-methylstyrene), poly(o-methylstyrene), poly(m- methylstyrene), poly(p-methylstyrene), poly(dimethylphenylene oxide), polyurethane, polyvinyl chloride, or mixtures thereof.

[0028] The end use of the surface or desired properties of the surface will dictate the type of hydrophilic polymer, cross-linking agent, and optional activating agent. These choices can be made by the person of skill in the art, given the disclosure herein.

[0029] Hydrophilic polymers suitable for use in the disclosed methods include, but are not limited to, polyacrylic acids and other polymers having accessible carboxylic acid functional groups, amino-functionalized polymers, hydroxyl-functionalized polymers, or polymers with functional groups that may react with a functionalized surface as described above. Nonlimiting examples of hydrophilic polymers suitable for use in the present methods include polyacrylic acid, polycarboxylate, polysulfonate, polyphosphate, polyphosphonate, polyethylene glycol, polyvinyl alcohol, polyvinyl amines, polylactates, and the like. In some cases, the polymer used is hydrophilic and/or water-soluble. The solubility of the polymer may be controlled by its molecular weight. Typically, the molecular weight is about 10 kDa to about 500 kDa. In some cases, the molecular weight of the polymer is less than 250,000 Da. In other cases, the molecular weight is less than 200,000 Da.

[0030] The polymeric acids typically are prepared from ethylenically unsaturated monomers having at least one hydrophilic moiety, such as carboxyl, carboxylic acid anhydride, sulfonic acid, and sulfate. The polymeric acid can contain a comonomer, such as styrene, an alkene, and/or a partially or fully fluorinated alkene or styrene to increase the hydrophobicity of the polymeric acid.

[0031] Examples of monomers used to prepare the polymeric organic acid include, but are not limited to:

(a) Carboxyl group-containing monomers, e.g., monoethylenically unsaturated mono- or polycarboxylic acids, such as acrylic acid, methacrylic acid, maleic acid, fumaric acid, crotonic acid, sorbic acid, itaconic acid, ethacrylic acid, α-chloroacrylic acid, α-cyanoacrylic acid, β-methlacrylic acid (crotonic acid), α-phenylacrylic acid, β-acryloxypropionic acid, sorbic acid, α-chlorosorbic acid, angelic acid, cinnamic acid, p-chlorocinnamic acid, β- stearylacrylic acid, citraconic acid, mesaconic acid, glutaconic acid, aconitic acid, tricarboxyethylene, and cinnamic acid;

(b) Carboxylic acid anhydride group-containing monomers, e.g., monoethylenically unsaturated polycarboxylic acid anhydrides, such as maleic anhydride; and

(c) Sulfonic acid group-containing monomers, e.g., aliphatic or aromatic vinyl sulfonic acids, such as vinylsulfonic acid, allylsulfonic acid, vinyltoluenesulfonic acid, styrenesulfonic acid, sulfoethyl (meth)acrylate, 2-acrylamido-2-methylpropane sulfonic acid, sulfopropyl (meth)acrylate, and 2-hydroxy-3-(meth)acryloxy propyl sulfonic acid.

[0032] The polymeric acid can contain other copolymerizable units, i.e., other monoethylenically unsaturated comonomers, well known in the art, as long as the polymer is substantially, i.e., at least 10%, and preferably at least 25%, acid group containing monomer units. To achieve the full advantage of the present invention, the polymeric acid contains at least 50%, and more preferably, at least 75%, and up to 100%, acid group containing monomer units. The other copolymerizable units, for example, can be styrene, an alkene, an alkyl acrylate, an alkyl methacrylate, and/or a partially or fully fluorinated alkene or styrene.

[0033] Suitable pairings of functionalized surface, polymeric group, cross-linking agent, and target molecule will be apparent to one of skill in the art in view of the present disclosure. For example, a surface functionalized with carboxylic acids may be reacted with hydrophilic polymers having amines, hydroxyl, and/or thiol groups, with cross-linking agents containing dicarboxylic acids, esters, or anhydrides, and further reacted with target molecules having functionality capable of reacting with amines, hydroxyl, and/or thiol groups, such as carboxylic acids, esters, anhydrides and the like.

[0034] The disclosed methods may include mixing a cross-linking agent with the polymer and the surface to provide modified surfaces which can be suitable for detection assays. Nonlimiting examples of cross-linking agents include diamines of various molecular weights, diols of various molecular weights, thiols of various molecular weights, and dicarboxylic acids of various molecular weights. The molecular weight of the cross-linking agent may play a role in the end properties of the modified surface by changing the contact angle of the available reactive sites on the modified surface for the target molecule with which to react. The difference in contact angle may be reflected in the resulting size of the spot resulting from the polymer attaching to the surface. The molecular weight of the crosslinking agent may also play a role in the amount of possible loading of the target molecule to the modified surface. Specific diamines that may be used in methods disclosed herein include hydrazine, C_2-15 diamines, and specifically, C₈, and Ci₂ diamines, where the number refers to the length of the alkyl chain separating the amine functional groups. C₈ refers to a diamine with an eight carbon alkyl group separating the two amine functional groups (e.g., 1,8- diaminooctane); and Ci₂ refers to a diamine with a twelve carbon alkyl group separating the two amine functionalities (e.g., 1,12-diaminododecane).

[0035] A coupling reagent optionally can be added to facilitate the reaction between the functional group on the surface (e.g., an aminated surface having amines or a hydroxylated surface having alcohols) and the carboxylic acid of the polymer. Such coupling agents may include those commonly used in peptide chemistry such as HOAt (l-hydroxy-7- azabenzotriazole), HOBt (1-hydroxybenzotriazole), HATU (O-(7-aza-benzotriazol-l-yl)- N,N,N',N'-tetramethyluronium hexafluorophosphate), HBTU (0-(benzotriazol-l-yl)- N,N,N',N'-tetramethyluronium hexafluorophosphate), EDC (N-(3-Dimethylaminopropyi)-N'- ethylcarbodiimide), DCC (dicyclohexylcarbodiimide), and DIC (diisopropylcarbodiimide).

[0036] In some cases, the present methods utilize agents that modify free carboxylic acid sites of the hydrophilic polymer to form more reactive functional groups, e.g., activated sites. Nonlimiting examples of such activating agents include N-hydroxysuccinimide (NHS) which can transform carboxylic acids to N-hydroxy succinimide esters (NOS). Additionally, the coupling agents listed above can be used as activating agents.

[0037] The activated sites then can be used to attach target molecules. Suitable target molecules include polypeptides and proteins, nucleic acids, and small molecules. Any suitable target molecule will have a suitable functional group to covalently bond to the activated site of the modified surfaces as disclosed herein. Typically, a nucleic acid or protein or polypeptide can bind to an activated ester (e.g., a NOS). Small molecules having an alcohol functional group, an amine functional group, or a thiol functional group can also form a covalent bond to an activated ester at the activated site.

[0038] As used herein, the term "nucleic acid" refers to an oligonucleotide or polymeric compound comprising bases of DNA, RNA, or combinations thereof. Alternatively, a polynucleotide is referred to herein as a "oligomer", i.e., a DNA oligomer or RNA oligomer. Non-limiting examples of bases that comprise a polynucleotide used in the present invention include adenosine, guanosine, cytosine, thymidine, inosine, cytidine, uridine, pyrimidine, uracil, thymine, purine, methylcytosine, 5-hydroxymethylcytosine, 2-methyladenine, 1- methylguanine, 2,6-diaminopurine, 2-amino-6-chloropurine, 2,6-dichloropurine, 6- thioguanine, 6-iodopurine, 6-chloropurine, 8-azaadenine, allopurine, isoguanine, orotidine, xanthosine, xanthine, hypoxanthine, 1 ,2-diaminopurine, pseudouridine, C-5-propyne, isocytosine, isoguanine, 2-thiopyrimidine, rhodamines, benzimidazoles, ethidiums, propidiums, anthracyclines, mithramycins, acridines, actinomycins, merocyanines, coumarins, pyrenes, chrysenes, stilbenes, anthracenes, naphthalenes, salicylic acids, benzofurans, indodicarbocyanines, fluorescamine, psoralen, and other commercially available or synthesized bases.

[0039] The terms "polypeptide" and "protein" are used interchangeably herein. Polypeptides typically comprise 5 to about 600 amino acids, but can be any length or any protein of interest. Amino acids can be either natural or synthesized.

[0040] The disclosed methods provide modified surfaces which can be used in a variety of assays, including detection assays. Such detection assays are described in, e.g., U.S. Patent Nos. 7,098,320; 7,063,946; 6,986,989; 6,974,669; 6,969,761; 6,962,786; 6,861,221; 6,858,387; 6,828,432; 6,827,979; 6,812,334; 6,777,186; 6,773,884; 6,767,702; 6,750,016; 6,730,269; 6,720,411; 6,720,147; 6,709,825; 6,682,895; 6,673,548; 6,645,721; 6,582,921; 6,506,564; 6,495,324; and 6,361,944, each of which is incorporated in its entirety by reference herein.

[0041] In embodiments where the surface is a glass surface, the surface can be selectively modified at multiple sites, so as to provide a surface having isolated modified sites suitable for use in a detection assay. Each site can optionally be modified with different target molecules so as to assess each individually in a single detection assay. The sites are isolated from one another so as to ensure that no site influences or falsely indicates an adjacent site has activity, inhibition, or some other type of interaction of interest in the detection assay.

EXAMPLES

[0042] The following examples are provided to illustrate the disclosed method, but are not intended to limit the scope thereof.

[0043] A schematic of a general method is laid out in FIG. 1. A glass slide is modified with an aminoalkylsilane to provide an animated surface. This surface then is treated with a cocktail of reagents, including a random polyacrylic acid co-acrylamide polymer with an average molecular weight of about 200,000 Da, a coupling reagent (EDC), a modifying reagent (NHS), and a cross-linking reagent (diamine). The resulting slide is a modified surface having the random polyacrylic acid co-acrylamide polymer attached via the amine functionality on the surface. Some of carboxylic acid functional groups on the polymer react with the NHS to provide NOS functional groups. Other carboxylic acid functional groups on the polymer react with the diamine cross linking agent to provide a more rigid structure. [0044] The modified surface is suitable for treatment with any analyte having a reactive group compatible with the reactive group on the polymer. As seen in FIG. 2, the NOS functional groups on the polymer react with a biomolecule having a free amine to covalently attach the biomolecule to the modified surface.

[0045] Preparation of Modified Surfaces: Glass slides were washed in a 1 % solution of sodium hydroxide containing 0.01% sodium dodecyl sulfate (SDS) for 30 minutes (min). The slides then were washed with distilled water and placed in a bath containing a 1% solution of aminopropyltrimethoxysilane for two hours at room temperature. The slides then were washed with distilled water and heated at 100⁰C for 10 min. A 50 mL polymer "cocktail" solution was prepared in 0.1M morpholinoethylsulfonic acid (MES), 1% poly(acrylamide-co-acrylic acid) partial sodium salt acrylamide 20 wt% (average MW 20OkDa), 750 mg N-hydroxysuccinimide, 500 mg EDC, and 600 μmol of a diamine. Any diamine, mixture of diamines, or salts of diamines (e.g., amine hydrochlorides) may be used. The slides then were placed in a flat polyethylene dish and the cocktail poured over them and allowed to react for one hour with 80 revolutions per minute (rpm) shaking at room temperature. The slides then were washed three times with distilled water, dried, and passivated using acetic anhydride and 10% methylimidazole in a tetrahydrofuran (THF)/pyridine mixture (50 mL) for 2 min. The slides then were washed with THF two times, acetonitrile two times, and dried under nitrogen. The slides then were placed in a dessicator until use.

[0046] Polymer Surface Results with Silver Amplification: Three different surfaces were prepared in the method outlined in the preparation example above, wherein the diamine was varied from a C₃, C₈, and C₁₂ diamine. The resulting surfaces then were printed with HIV amino capture probe (3'NH₂-AAA AAA AAAA ACG TAG GTC CAG TAC) (SEQ. ID. NO.: 1), and allowed to stand in a 50% humidity chamber overnight. The surfaces then were washed with 0.2% SDS prior to experiments for 5 minutes (min) to remove any unattached capture probes. A synthetic target (5' TGC ATC CAG GTC ATG TTA TTC CAA ATA TCT TCT) (SEQ. ID. NO.: 2) for the HIV amino capture probe was then incubated for 30 min at 37°C in hybridization buffer containing SDS, Tween 20, 2.5x sodium chloride-sodium citrate (SSC), and 10% formamide at 1 nM gold probe concentration. Gold nanoparticles (13 nm) modified with a nucleotide sequence (5' SH-AAA AAA AAAA AGA AGA TAT TTG GAA TAA) (SEQ. ID. NO.: 3) were used for detection. After incubation, the surfaces then underwent silver development (see, e.g., Mirkin et al., Science.2%9: 1757 (2000)) at room temperature for 5 min, and then were scanned using a Verigene ID scanner. [0047] FlG. 3 shows the results of the detection experiment using surfaces that were modified with C₃ diamine cross-linkers. The amount of synthetic target was varied from 500 fM up to 100 pM, with all concentrations of synthetic successfully detected. FIG. 4 shows similar results with the C₈ diamine cross-linker and FIG. 5 with the C₁₂ diamine cross-linker.

[0048] Polymer Surface Results with Fluorescein Probe (FITC): Surfaces modified using C₃ and C₈ diamine cross-linkers were prepared in the manner as outlined in the preparation example above. The surfaces were printed with HTV amino capture probe (3'NH₂-AAA AAA AAAA -ACG TAG GTC CAG TAC) (SEQ. ID. NO.: 1) and allowed to stand in a 50% humidity chamber overnight. The surfaces were washed with a 0.2% SDS solution prior to the experiments for 5 minutes prior to hybridization to remove any unbound capture probes. The surfaces were then treated with InM synthetic target (5' TGC ATC CAG GTC ATG TTA TTC CAA ATA TCT TCT) (SEQ. ID. NO.: 2) for 30 min at 37°C in a hybridization buffer containing SDS, Tween 20, 2.5x SCC and 10% formamide at 100 nM fluorescent probe (5'FITC-AAA AAA AAAA -AGA AGA TAT TTG GAA TAA) (SEQ. ID. NO.: 4) concentration. Concentrations less than 1 nM were not detected under these conditions. The surfaces were then analyzed using a Leica TCS SP2 with a 488 Argon laser line excitation and a 10x objective. The resulting images are seen in FIG. 6 for both C₃ and C₈ modified surfaces. These images clearly show the difference in spot size which may be derived from the different diamine cross-linkers.

[0049] Modified Surfaces Containing Electrodes: Surfaces containing electrodes (electrode chip was made of gold on silicon/silicon oxide (oxide layer 500 nm)) were aminated using aminopropyltrimethoxysilane in a manner similar to that outlined in the preparation of glass slide surfaces. The animation of the surface was followed by immobilization of a polymer cocktail (as described in the glass surface example). Amino oligo-nucleotides were reacted with the immobilized polymers in a phosphate buffer pH 7.8, then allowed to stand at 50% humidity for 4 hours. The slides then were washed and hybridized in a hybridization buffer containing 13 nm gold particles in the presence and absence of a target nucleotide. The electrode that was treated with the target nucleotide has a deposition of silver, while the electrode that was not treated with the target nucleotide has no silver deposition, after silver staining.

[0050] The present methods are not dependent upon the nucleotide sequences, targets, or probes used. It is envisioned that any nucleotide sequence having the appropriate functionality are suitable for use with the surfaces prepared by the methods disclosed herein.

Claims

What is Claimed:

1. A method for modifying a surface comprising the steps of: a) contacting (1) an unmodified surface with (2) a compound having an amino functional group and a second functional group capable of covalently bonding to said unmodified surface, to form an aminated surface; b) contacting the aminated surface with (1) a hydrophilic polymer, (2) a cross- linking reagent, and optionally (3) a coupling reagent, to form the modified surface, wherein the hydrophilic polymer comprises a plurality of carboxylic acid or carboxylic acid ester functional groups, or a mixture thereof.

2. The method of claim 1, wherein the cross-linking agent comprises a compound having at least two functional groups independently selected from the group consisting of -OH, -SH, and -NH₂.

3. The method of claim 1, wherein the cross-linking agent comprises a diamine.

4. The method of claim 3, wherein the diamine is selected from the group consisting of hydrazine, a linear or branched C_2-15 diamine, and mixtures thereof.

5. The method of claim 1 , wherein the hydrophilic polymer is selected from the group consisting of polyacrylic acid, polycarboxylate, polysulfonate, polyphosphate, polyphosphonate, polyethylene glycol, polyvinyl alcohol, and mixtures thereof.

6. The method of claim 1 , wherein the hydrophilic polymer has a molecular weight of about 10 kDa to about 500 kDa.

7. The method of claim 6, wherein the hydrophilic polymer has a molecular weight less than about 200 kDa.

8. The method of claim 1 , wherein the surface is hydrophobic prior to modification.

9. The method of claim 8, wherein the surface is a hydrophobic polymer surface.

10. The method of claim 9, wherein the hydrophobic polymer is selected from the group consisting of polystyrene, polyethylene, polybutylene, polypropylene, polymerized mixed olefins, polyterpene, polyisoprene, polyvinyltoluene, poly(α-methylstyrene), poly(o- methylstyrene), poly(τw-methylstyrene), poly(p-methylstyrene), poly(dimethylphenylene oxide), polyurethane, polyvinyl chloride, and mixtures thereof.

11. The method of claim 1 , wherein the surface is a glass surface.

12. The method of claim 11 , wherein the glass surface is modified at isolated multiple sites.

13. The method of claim 1, wherein the surface is a gold nanoparticle or a gold microparticle.

14. The method of claim 13, wherein the second functional group comprises a thiol or a sulfide.

15. The method of claim 1 , wherein the surface comprises a hydrophobic polymer.

16. The method of claim 1, wherein step (b) further comprises contacting with an activating agent, wherein the activating agent reacts with at least one carboxylic acid or carboxylic ester of the hydrophilic polymer to form at least one activated site.

17. The method of claim 16, wherein the activating agent comprises N-hydroxy succinimide.

18. The method of claim 16, further comprising: c) contacting a target molecule and the resulting surface of step (b), wherein the target molecule covalently binds to the activated site.

19. The method of claim 18, wherein the target molecule is a polypeptide or a nucleic acid.

20. A method for modifying a surface comprising the steps of: a) contacting (1) an unmodified surface with (2) a compound having a carboxylic acid functional group and a second functional group capable of covalently bonding to said unmodified surface, to form a carboxylated surface; b) contacting the carboxylated surface with (1) a hydrophilic polymer, (2) a cross-linking reagent, and optionally (3) a coupling reagent, to form the modified surface, wherein the hydrophilic polymer comprises a plurality of amine, alcohol, or thiol functional groups, or a mixture thereof.

21. The method of claim 20, wherein the cross-linking agent comprises a compound having at least two functional groups independently selected from the group consisting of -CO₂H and -CO₂Ci_-3alkyl, or mixtures thereof.

22. The method of claim 20, wherein the cross-linking agent comprises a dicarboxylic acid, dicarboxylic ester, or anhydride.

23. The method of claim 20, wherein the dicarboxylic acid, dicarboxylic ester, or anhydride comprises HO₂C-C_0-15alkylCO₂H, Ci-₃alkylO₂C-C_0-15alkylCO₂C_1-3alkyl, maleic anhydride, succinic anhydride, phthalic anhydride, or mixtures thereof.

24. The method of claim 20, wherein the hydrophilic polymer comprises a polyamine, polyvinyl alcohol, or mixtures thereof.

25. The method of claim 20, wherein the hydrophilic polymer has a molecular weight of about 10 kDa to about 500 kDa.

26. The method of claim 25, wherein the hydrophilic polymer has a molecular weight less than about 200 kDa.

27. The method of claim 20, wherein the surface is hydrophobic prior to modification.

28. The method of claim 27, wherein the surface is a hydrophobic polymer surface.

29. The method of claim 20, wherein step (b) further comprises (1) optionally contacting with an activating agent, wherein the activating agent reacts with at least one amine, alcohol, or thiol of the hydrophilic polymer to form at least one activated site, and (2) contacting a target molecule, wherein the target molecule covalently binds to the activated site or to the hydrophilic polymer.

30. The method of claim 29, wherein the target molecule is a polypeptide or a nucleic acid.

31. A method for modifying a surface comprising the steps of: a) contacting (1) an unmodified surface with (2) a compound having a hydroxyl functional group and a second functional group capable of covalently bonding to said unmodified surface, to form a hydroxylated surface; b) contacting the hydroxylated surface with (1) a hydrophilic polymer, (2) a cross-linking reagent, and optionally (3) a coupling reagent, to form the modified surface, wherein the hydrophilic polymer comprises a plurality of carboxylic acid or carboxylic acid ester functional groups, or a mixture thereof.

32. The method of claim 31, wherein step (b) further comprises (1) optionally contacting with an activating agent, wherein the activating agent reacts with at least one carboxylic acid or carboxylic acid ester functional group of the hydrophilic polymer to form at least one activated site, and (2) contacting a target molecule, wherein the target molecule covalently binds to the activated site or to the hydrophilic polymer.

33. A modified surface prepared according to the method of claim 1.

34. A modified surface prepared according to the method of claim 18.

35. A modified surface prepared according to the method of claim 20.

36. A modified surface prepared according to the method of claim 29.

37. A modified surface prepared according to the method of claim 31.

38. A modified surface prepared according to the method of claim 32.