Functional surface coatings and methods for the preparation thereof

X.

<img img-format="tif" img-content="drawing" file="IL159556AD00021.tif" id="idf0001" />

FUNCTIONAL SURFACE COATINGS AND METHODS FOR THEPREPARATION THEREOF onnnb rnu&gt;vn (mown ”i£ps

<img img-format="tif" img-content="drawing" file="IL159556AD00022.tif" id="idf0002" />

Field of the Invention

This invention relates generally to the field of surface coatings that impart 10 desirable chemical, physical, and biological properties to a substrate material. In particular, this invention relates to surface chemistries designed to inhibit non-specificadsorption or binding of molecules, particles, or cells to a surface while also providing ameans for immobilizing or entrapping specific molecules, capture agents, drugs, particles,or cells to or within that same surface. This invention further relates to coatings for 15 application to a variety of chemically distinct substrate materials.

Background of the invention

Controlling the interactions between naturally derived or synthetic materials andbiomolecule-containing fluids is of increasing importance in a variety of fields. Forexample, biomolecule interactions with a variety of substrate materials, i.e., surfaces, are 20 centra] to numerous analytical systems, including immunodiagnostics, gene and proteinmicroarrays, and microfluidic “lab-on-a-chip” devices. Analytical techniques such ascapillary electrophoresis (CE), surface plasmon resonance (SPR), and quartz crystalmicrobalance (QCM) also intimately depend on biomolecule-surface interactions.Performance of biomedical devices including cardiovascular replacements (e.g., catheters, 25 valves, stents), contact and intraocular lenses, shunts, filters, diaphragms, pumps, membranes, drug delivery devices and surgical components also depend on control ofbiomolecule-surface interactions. Maritime surfaces, including heat exchange units, ship-board components (e.g., hulls, superstructure), installations (pump derricks), docks, andenvironmentally exposed instruments also require control of biomolecule.deposition 30 (algae, fungus, microbial exudates) on their surfaces.

Of major common concern within these fields is the level of non-specific biomolecule (e.gl, protein, organismal, nucleic acid) binding to target and devicesubstrates, hi non-sensing or non-diagnostic applications, this concern general focuses onsurface-induced biofouling (deposition of biological material that compromises function).

This is particularly problematic in biomedical devices and maritime applications. Inmedical, food, and environmental sensing and diagnostics, specificity, signal-to-noiseratios, and detection limits of a target analyte (e.g., polysaccharides, nucleic acid, drug,peptide or protein), often at limited concentrations in a milieu of non-target biomolecules(e.g., serum proteins), are limited by surface non-specific non-target (e.g., protein)binding on the substrate. Reduction or elimination of this non-specific binding noise willimprove device performance, and enhance signal-to-noise ratios, detection sensitivity andspecificity of many analytical systems.

Beyond analytical systems, the synthetic material-biological interface is central tothe proper function of many biomedical devices. For example, non-specific binding ofproteins at medical implant surfaces is believed to be at least partially responsible fortriggering the foreign body response, which in turn can lead to device failure or rejection.This biofouling is also blamed for device infection incidence, thrombosis, and sensordeterioration over time in vivo. Alternatively, it has been hypothesized that implantsurfaces that interact with target biomolecules in a specific manner can be used tostimulate natural healing, avoiding many adverse reactions from biofouling. In addition,numerous non-analytical and non-medical applications require direct control of surface-biological fluid interactions. Examples include non-fouling paints and anti-microbialcoatings for industrial equipment, colloid and mineral handling systems, cooling systems,marine structures, and bioreactors used for production of biological products in vitrousing biological components.

Improvements in these important fields all would benefit from development ofimproved surface chemistries where specific target binding, tethering or entrapment canbe controlled versus non-specific deposition of undesired components. A corerequirement of these surface chemistries is that the synthetic surfaces show reduced orlimited non-specific binding (NSB) to non-targets, biomolecules, particles, or cells. NSBoccurs through a variety of basic molecular-level adhesion mechanisms, including allcombinations of electrostatic, hydration, hydrophobic, acid-base, dispersive and hydrogenbonding interactions. Soluble proteins, for example, generally ubiquitously anduniversally adsorb to a surface through combinations of these non-specific interactions,creating an adherent layer of biological material difficult to remove (a critical step inbiofouling). Protein NSB often results in protein denaturation (loss of native structure) onsurfaces that can prompt a cascade of additional NSB events (further adsorption of otherproteins, cells, micro-organisms, etc., to the exposed interior of the denatured protein). 2

This biofouling NSB cascade produces undesired infection, coagulation, inflammatoryresponses for biomedical implants, reduced signal-noise ratios for diagnostic assays andsensors, corrosion and deterioration for maritime and environmentally exposed structures,turbulence and reduced propulsion efficiency for maritime shipping, and undesiredpressure drops and flow properties in capillaries, tubes and microfluidic chambers.Inhibition of protein and biomolecule NSB is thus an important performance feature inthe design of improved synthetic materials that contact, function with, and operate inbiological fluids.

While NSB to surfaces is most often undesirable, specific capture of designatedbiomolecules, particles, drugs or cells by binding at a target surface is often desirable.Examples include the specific binding of bioactive antibodies on a surface forimmunoassay applications, specific binding of nucleic acid primers on a surface forpolymerase chain reaction (PCR) or genetic assays (microarrays), and specific binding ofgrowth factors or antibiotics to surfaces to promote or hinder cell growth, respectively.The goal of such specific binding to a surface is to bind only one designated type ofmolecule, particle, or cell, to the target surface and to do so in a manner that preserves therecognition activity and native structure and function of the specifically bound molecule,particle or cell.

Thus, functional surface chemistries are needed that (1) inhibit non-specificbinding of unwanted molecules, particles, or cells to a treated surface; (2) inhibit non-specific binding of unwanted molecules, particles, or cells to a treated surface whilepromoting specific biomolecule, particle, or cell binding, to that same surface; or (3) thatinhibit non-specific binding of unwanted molecules, particles, or cells to a treated surfacewhile promoting specific and functionally or biologically active biomolecule, particle, orcell binding, to that same surface.

Several strategies have been employed to create surfaces with these desired lownon-specific binding properties. These strategies typically involve selection of coatingchemistry or surface functional groups that exhibit low NSB and then fixing them to anunderlying substrate. The fixing step may he based on physical adsorption or on directcovalent linkage (chemical coupling). From a performance perspective, the layer mustexhibit robust bonding to the underlying support while maintaining low-NSB propertiesto the environment.

Surface coating strategies based on synthetic polymers have been the focus fordevelopment of most non-fouling coating applications. A. S. Hoffman, “Non-Fouling 3

Surface Technologies,” Journal of Biomaterials Science: Polymer Edition 10, no. 10(1999): 1011-1014; Poly(ethylene glycol): Chemistry and Biological Applications, ACSSymposium Series 680, J. Milton Harris and Samuel Zalipsky, Eds., American ChemicalSociety, 1997. Hydrophilic, polar, electrically neutral polymers such as polyethylene 5 glycol (PEG) derivatives have received significant attention because of their long-acknowledged abilities to reduce protein NSB from solutions in coated formats. Thechallenge has been to effectively fix these and other similar water-soluble polymers touseful substrate materials at densities sufficient to impart effective NSB throughcontiguous coating. One approach has been to physically adsorb hydrophilic- 10 hydrophobic block copolymers to hydrophobic supports. Hydrophobic interactionsbetween the substrate and hydrophobic polymer block are sufficient to attach coatingpolymer molecules to the substrate while presenting the hydrophilic, low-NSB block tothe aqueous surroundings. U.S. Patents 5,075,400 and 6,093,559.’ Problems with thesephysically adsorbed films, however, include reversible polymer desorption, particularly in 15 challenging liquid environments (e.g., high salt, non-neutral pH, elevated temperature,shearing flow conditions, etc.), and the ability to achieve reproducible adsorbed densitieseffective to limit NSB .

Another approach to creating surfaces with low non-specific binding propertiesrelates to the covalent coupling of low-NSB coating polymers to the target surface. PEG, 20 for example, can be covalently grafted to substrates through reactive end groups introduced to the PEG molecule. U.S. Patent 5,512,329. Azido chemistry and quinonechemistry are two photo-reactive end group examples for such fixation to polymersubstrates. A disadvantage of this approach is that it is confined to coating particulartypes of polymer supports where this coupling chemistry is effective and available. 25 Attachment to interesting inorganic substrates such as glass or metals requires someintermediate substrate attachment layer, which adds to the cost, time consumption, andoverall effectiveness of the coating procedure. One particular example of this procedurerelies on PEG-silanes used to create coatings on active metal oxide surfaces (glass, silica,titania, alumina and others).. In this system, a PEG molecule is derivatized with 30 alkoxysilane or chlorosilane terminal reactive groups. Hydrolysis and chemical condensation of the reactive silanes presumably serve to anchor the PEG molecules tooxide substrates such as glass or silicon oxide with exposed surface silanol groups.Despite their initial promise, PEG-silane surfaces are difficult to reproduce practically. Itis believed that the terminal alkyl silane reactive groups hydrolyze in solution and then 4 react with each other in the bulk solution, prior to attachment to the substrate, yieldspoorly defined films (reactive groups cross-react with each other rather than to anchor thePEG-silane to the surface) allowing for a partial physically adsorbed polymer coatingmixed with some fraction of chemical attachment. Control of this for reliable coating andlow NSB is difficult.

An alternative to forming a polymer film for blocking non-specific binding on asubstrate is to inhibit the non-specific binding to the substrate with a biomolecule-blocking step (masking). The most common practice in this respect is to pre-adsorbsubstrates with soluble bovine serum albumin (BSA), casein, or serum. These proteins(or proteins in the serum) adsorb strongly to most surfaces from aqueous solution,providing an adsorbed protein layer that minimizes subsequent non-specific binding ofother biomolecules. While such protein blocking has proven to be functionally effective,the blocking step is time consuming, labor intensive, and subject to reproducibilityproblems, bi addition, there is a general desire to move away from the use of animal andserum-derived products in surface chemistry applications for safety reasons.

Thus a perceived improvement in the art is a solid-phase surface coating capableof consistently strong, direct and effective attachment to a variety of substrate materials.The surface should reliably exhibit low non-specific binding properties and should beeasy to use (i.e, should require no blocking steps). Ideally, the surface chemistry shouldalso provide a means for immobilizing chemically reactive coupler or functional groupson the surface, which in turn exhibit strong specific attachment properties for desiredtarget immobilization onto surfaces. Against this backdrop of technical-challenges andcurrent limitations in the field, the present invention has been developed.

Summary of the Invention

Embodiments of the present invention are directed at novel surface chemistriesand methods of preparing functional solid-phase coatings and surfaces with improvedproperties both limiting NSB and enabling specific binding. The present inventionprovides for robust coating bonding over various substrates, including oxide, metal,composite, ceramic, and polymer substrates while simultaneously imparting surfaceproperties that substantially inhibit non-specific binding of solutes from external milieu.

Without wishing to be bound to a particular theory, it is considered that theinteraction between the various components of the surface chemistry, described in moredetail below, provide the strengths and benefits of a novel surface coating with robustattachment to a variety of chemically distinct solid phase substrates. The chemistry of the coating substantially reduces dependence upon a single attachment mechanism (i.e.,silane coupling, physical adsorption, photoreaction, chemical attachment, etc.). Instead,the multifunctional nature of the coating chemistry provides multiple likely attachmentmechanisms, including covalent attachment to oxide, ceramic and polymer substrates,crosslinking within the coating, and adsorption via hydrophobic interactions, acid-basechemistry, hydrogen bonding, etc., which act in combination or synergistically to stabilizethe coating on the substrate and reliably present the desired surface properties.Importantly, chemical cross-linking within the applied coating itself is believed toaugment the coating cohesive character, coating-substrate interactions, and add to therobustness of substrate attachment.

The present invention promotes formation of a functional surface coating. Theterm “functional” is used herein to describe the ability to perform a specific chemical oranalytical task. For example, one class of functional surfaces can provide specific affinitybinding or ligand-receptor binding capabilities with designated solutes. For example, asurface can be created that presents biotin groups consistently at the surface. Biotingroups exhibit a highly specific, high affinity binding interaction with streptavidin proteinmolecules and their mutants. The functional surface in tliis example, therefore, is abiotinylated, low background NSB coating where function is defined by the ability tospecifically bind streptavidin.

In another embodiment ofthe present invention, the functional surfaces aredesigned to inhibit all molecular binding interactions to the coating. In this case, thespecific functional task is to be interfacially inert. A manifestation of such “inert”function could be a surface that presents effective densities of methoxy-capped ethyleneoxide oligomers or polymers. Such surface groups generally do not form covalentlinkages, generally do not show affinity binding properties to proteins, cells, or particles,and in general resist adsorption of biomolecules and other soluble species. In the contextof the present invention, these inert groups are incorporated directly into the underlyinglow-background NSB coating invention.

The present invention embodies a coating architecture comprising designatedfunctional surface groups (e.g., methoxy-PEG or biotin) integrated with a cross-linked,low-NSB coatable matrix. The surface functional groups are covalently coupled to thecoating matrix through spacer molecules selected to provide conformational flexibilityand to be inert to non-specific adsorption. The low-NSB matrix comprises these spacersand other so-called matrix-forming molecules that have been cross-linked into the 6 coatable film. Exemplary matrix-forming molecules include polysaccharides (dextran),ethylene oxide-containing oligomers (linear, block, dendritic or star polymer forms),block co-polymers (Poloxamer®, Pluronics®), and non-ionic surfactants (Tween®,Triton®). The coating architecture is believed to rely on a combination of both physical 5 and chemical cross-linking mechanisms. In addition to physical entanglement of largemolecules in the matrix, hetero- or bi-functional, reactive cross-linker molecules may alsobe included and chemically activated in the coatings. Covalent cross-linking is believedto occur both within the coating matrix and to the substrate. An advantage of usinghetero- and bi-functional cross-linkers in this invention is that the coating can be 10 effectively applied and cross-linked to a variety of chemically distinct substrate materialswith or without specific surface attachment. The coating described in this inventionprovides robust attachment both within itself and also to various substrates.

The present invention provides methods of preparing a functional surface coatingusing the components with the described functions. The methods include providing a 15 substrate with an effective amount of an active component, providing an effective amountof a cross-linking component and providing an effective amount of a matrix-formingcomponent. The active component, the cross-linking component and the matrix-formingcomponent are incorporated onto the substrate, and integrated with each other to createthe stable, robust functional surface improvement. 20 The present invention also provides a functional surface for performance of immuno, genetic, microbial, cellular, particulate and other biochemical or target analytebinding assays. Thus, a non-specific binding matrix is attached to a substrate and anactive component is integrated into the non-specific binding matrix to impart thesecoating selectivities for various assay formats. 25 In addition, embodiments of the present invention provide functionally inert surface coatings for medical implant devices for effectively limiting host rejection of themedical implant device through attached ligands, incorporated drugs, and reduced NSBresponsible for biofouling of such devices in- or ex-vivo.

These and various other features as well as advantages which characterize the 30 invention will be apparent from a reading of the following detailed description and areview of the appended claims.

Brief Description of the DrawingsFig. 1 is a diagrammatic depiction of a functional surface, in accordance with one embodiment of the present invention. The coatable embodiment comprising an active ' 7 ' · . ' component, a cross-linking component, and a matrix-forming component areschematically depicted to represent their function within the integrated coating on thesubstrate. Note that a schematic representation of the components comprising the presentinvention is shown at top, a cross-sectional representation of possible coating architecture 5 is shown in the middle, and a three-dimensional representation of possible coatingarchitecture is shown at bottom.

Fig. 2 illustrates the non-specific binding of protein on biotin functional surfacesin accordance with one embodiment of the present invention on SiCtySi wafers, comparedto BSA- blocked SiCb/Si wafers. 10 Fig. 3 illustrates the non-specific binding of protein on thiol-reactive functional surfaces in accordance with one embodiment of the present invention on SiCVSi wafers,relative to BSA-blocked and bare SiOj/Si wafers.

Fig. 4 illustrates the non-specific binding of protein on BSA-blocked substratesand functional surfaces according to one embodiment of the present invention on various 15 plastic substrates.

Fig. 5 illustrates the non-specific binding of protein on a BSA-blocked substrateand functional surfaces in accordance with one embodiment of the present invention on ametal substrate, in particular a gold substrate.

Fig. 6 illustrates the non-specific binding of protein on functional surfaces in20 accordance with one embodiment of the present invention on glass substrate, BSA- blocked glass, and bare glass substrate.

Fig. 7 illustrates the non-specific binding of fibrinogen on bare glass, BSA-blocked glass, and functional surfaces in accordance with one embodiment of the presentinvention. 25 Fig. 8 illustrates the specific binding of streptavidin on biotin functional surfaces in accordance with one embodiment of the present invention.

Fig. 9 illustrates the specific binding of biotinylated horseradish peroxidase tostreptavidin functional surfaces in accordance with one embodiment of the presentinvention on glass substrates. 30 Fig. 10 illustrates the specific binding of thiolated streptavidin to thiol-reactive functional surfaces in accordance with one embodiment of the present invention onplastic substrates. Each concentration was run in triplicate and error bars represent onestandard deviation in the set. 8

Fig. 11 illustrates the specific binding of streptavidin to amine-reactive functionalsurfaces in accordance with one embodiment of the present invention on primed glasssubstrates. Each concentration was run in triplicate and error bars represent one standarddeviation.

Fig. 12 illustrates the specific binding of biotinylated antibody to a streptavidinfunctional surface in accordance with one embodiment of the present invention on glasssubstrate.

Figs. 13A and B illustrate the improved assay sensitivity for staphylococcalenterotoxin B for a functional surface according to one embodiment of the presentinvention. Note that Fig. 13A shows optical density at 450 nm and Fig. 13B showsmembrane thickness.

Figs. 14A, B and C illustrate the use of one embodiment of the present inventionin an oligonucleotide microarray application, and specifically illustrates the use ofoligonucleotides with amine-reactive functional surfaces in comparison to state-of-the-artcommercial polymer and silane chemistries. Fig. 14A shows fluorescence detection ofCy 5 Streptavidin bound to 3’ biotin labeled oligonucleotide. Signal intensities arereported in relative fluorescence units. Data were obtained from three representative mnsand error bars represent one standard deviation. Fig. 14B shows fluorescence backgrounddetermination where noise levels are reported in relative fluorescence units. Dataobtained from one slide and error bars represent one standard deviation. Fig. 14C showsthe resulting signal-to-noise ratios (S/N) for the coating chemistry compared to thepolymer and silane slide chemistries. One slide represented per data points.

Fig. 15 illustrates the use of one embodiment of the present invention in a proteinmicroarray application, specifically illustrating the use of streptavidin functional surfacesfor selective capture of biotinylated antibodies on glass substrate.

Fig. 16 illustrates the non-specific binding of various coated surfacesdemonstrating synergistic effects of the functional surface according to one embodimentof the present invention.

Figs. 17A and B illustrates the use of different matrix-forming components inseveral embodiments of the present invention that show low non-specific protein bindingand high specific protein attachment.

Figs. 18A and B illustrates the inhibition of fibroblast cell proliferation after threedays of culture on a coated surface embodiment of the present invention (A) relative to atissue culture polystyrene control (B). 9

<img img-format="tif" img-content="drawing" file="IL159556AD00023.tif" id="idf0003" />

159,556/2

Detailed Description of Preferred EmbodimentsThe features and other details of the preferred embodiments of the present invention will now be more particularly described. It is to be understood that theparticular embodiments of the invention are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed invarious embodiments without departing from the scope of the invention.

The present invention provides methods and compositions for the preparation ofmulti-component, multi-functional thin film coatings 100 applied to and stabilized upon avariety of substrates 102 (see Fig. 1). These coatings 100 substantially inhibit non- 10 specific binding or adsorption of non-target proteins, e.g., fibrinogen, undesired enzymesand antibodies, undesired nucleic acids, polysaccharides, particulate solutes, microbes,cells, colloids, and the like (collectively termed non-target analytes), to the coatedsubstrate. The present invention also describes such surfaces stabilized upon a variety ofsubstrates 102 that provide for very low non-specific binding and, if desired, high specific 15 binding and increased assay selectivity and sensitivity. A diagrammatic depiction of a multi-component, multi-functional surface coating 100 according to one embodiment of the present invention is shown in Fig. 1. Fig. 1illustrates the components of the method and apparatus of the present invention and theirbelieved interactions. As shown in Fig. 1, a substrate 102 is provided with a low non- 20 specific binding matrix 104 affixed upon the substrate 102 and an active component 106physically entangled and covalently bound within the applied non-specific binding matrix104. The methods, coatings, compositions, use and kits, according to preferredembodiments of the present invention, are described in more detail below.

Methods of the present invention are directed to the preparation of functional 25 surfaces 100 capable of either or both specific binding and reduced non-specific bindingto a target substrate 102. The coating method includes providing effective amounts of anactive component 106, a cross-linking component 108, and a matrix-forming component104. These three general components are incorporated onto the substrate, therebycreating and comprising the functional surface coating 100 and imparting the properties 30 described by the invention.

The term “functional surface” 100 is used herein to describe a surface thatemploys specific chemical groups to selectively bind desired target molecules, particles,cells, and the like (collectively called target analytes), from external milieu, and/or tocollectively reject undesired molecules, particles, cells, and the like (collectively called 10 non-target analytes), from the surface. By this definition, one class of functional surfaces 100 provides specific binding capabilities through the incorporation of specific functional groups 110 at the surface. An example is a biotinylated surface that exhibits high avidin binding activity (typically in the range of 300 to 500 ng/cm2 of avidin) and low NSB of□ other biologies, particulates, and solutes (typically in the range of 0.01 to 50 ng/cm").

The term “functional surface” is also used herein to describe chemical groups or surfacecompositions that exhibit inherently low protein or particulate binding or adsorption inthe range specified (typically around 0.01 to 50 ng/cm2). Thus, the “function” in thisclass of surfaces is to be substantially inert to protein, cell, microbe and particulateadsorption. An example of such inert functionality is a surface that presents methoxy-capped ethylene oxide oligomers or polymers. These inert surface groups 112 generallydo not form covalent linkages, generally do not show affinity binding properties, and ingeneral tend to resist adsorption of biomolecules, particulates, cells, microbes, and othersoluble species (non-target analytes).

For the purposes of this application, “effective amount” is defined as an amount ofa specified coating component that, when combined with the other coating components,yields a coating matrix that meets specified performance standards for the desiredapplication (e.g., assay or implant coating). These performance standards are defined forboth “low non-specific binding or adsorption” (“low-NSB”) and “highly selective specificbinding,” as described below.

For the purposes of this application, the terms “low non-specific binding,” “lownon-specific adsorption,” and “low-NSB” are defined relative to the current method ofbovine serum albumin (BSA) blocking, i.e., masking. BSA-blocking is a process inwhich the soluble serum protein, bovine serum albumin, is physically adsorbed fromsolution to a substrate material prior to the use of that substrate in a biological fluidsetting. This pre-adsorbed BSA layer serves to inhibit additional solute or particulateadsorption on the substrate. The BSA-blocking method represents a practical state-of-the-art process for the inhibition of non-specific binding or adsorption to a variety ofsubstrates, particularly in the context of biochemical assays. Thus, for the purposes of thepresent invention, a coating formulation that inhibits NSB in a manner similar to orsuperior to BSA-blocking, without the requirement of a BSA-preadsorption or anyequivalent blocking step, is herein considered to be a low-NSB surface.

The “highly selective specific binding” performance standard is also defined in acomparative manner. For the purposes of this application, a surface is considered to be it highly selective when the specific binding group incorporated in the surface is able toselectively bind target (analyte) species from a complex milieu, when a similar surfacelacking such specific binding groups shows little or no capacity to specifically hind targetspecies (analytes). For example, a biotinylated surface is said to be highly selective 5 relative to its non-biotinylated counterpart when significant, measurable quantities ofavidin or streptavidin are bound to the former and not to the latter under the same bindingconditions. A final performance standard is the combination of “no selectivity” and low-NSB.In this case, no binding or adsorption is desired, either specific or non-specific. Again, 10 performance is defined relative to a competing method such as BSA-blocking.

The incorporation or affixing step of the surface coat 100 onto a substrate 102 preferably comprises combining effective amounts of active component 106, a cross-linking component 108 and a matrix-forming component 104 into a coatable mixture.

This coatable mixture is applied to the substrate and cured to yield the stable, robust 15 surface. Coating mixtures may also include other non-covalently attached functionalgroups, for example streptavidin, which becomes entangled within the matrix.

The methods of the present invention preferably include combining the activecomponent, matrix-forming component, and cross-linking component in a carrier solventor solvent mixture. The range of component concentrations used depends on the 20 particular coating method chosen. Selection of solvent also depends on componentchemistry, the coating method and choice of substrate material, with factors such assolvent surface tension, solubility parameter, viscosity, and reactivity with the chosensubstrate influencing solvent selection.

Active Component For Use With The Present Invention: 25 Typically, embodiments of the active component 106 of the present invention include a functional group 110, a spacer group 114 and a binding group 116 (see Fig. 1).Note that where no specific binding is required, an inert functional group 112 can beinserted in place of the functional group 110.

In more detail, the term "active component" is intended to signify a bifunctional 30 reactive molecule that includes at least one reactive group (for example 116) at one end ofthe molecule and at least one functional group (for example 110 or 112) at either end ofthe molecule. The distance between the two groups can be varied by varying the lengthofthe spacer group 114 to provide the desired attributes as defined by the final productrequired by a person skilled in the art. As previously described, the active component 12 106 preferably includes specific chemical parts representing the functional group, spacer,and binding group. Specific definitions of these terms are provided below. The activecomponent 106 is preferably a combination of these groups; however, it is to beunderstood that the active component need not necessarily comprise three distinctlyseparate groups. It is possible that one group can act both as a functional group and aspacer group, for example. It is also possible, for example, that the functional group (forexample 110 or 112) and the reactive group 116 have the same chemical structure (e.g., ahomo bifunctional active component). The active component need only perform thefunction described with respect to each group of the active component as discussedbelow, with minimal parts needed to accomplish these objectives.

The term "functional group," 110 as used herein, is intended to include thosegroups that can generally interact with and form an association (both covalent and non-covalent) with target species external to the surface while generally not associating withnon-target species. The term “functional group” may also refer to those groups that areessentially inert to binding and adsorption of any or all species in external milieu, referredto as an inert functional group 112.

For surfaces designed for specific binding applications, functional.groups 110include but are not limited to chemically active groups, receptor or ligand groups, andchelators capable of selectively binding desired target species. More specific examples offunctional groups 110 include but are not limited to, for example, biotin, N-hydroxysuccinimide esters, nitrophenyl esters, carboxylates, vinyls, vinylsulfone, metal chelates,glutathione, streptavidin, nitrenes, acrylate groups, phenylboronic acid, pi trolotri aceticacid (NTA), imidodiacetate (IDA), salicylhydroxamic acid, hydroxyl groups (-OH),amine groups (-NH2), imine groups (-NHR) carboxylic acids (-COOH), aldehydes (-CHO), ketones (C=O), esters (-COOR), ethers (-C-O-C), amide groups (~CONH2), imides(-CONHR), cyanides (-CN), hydrazides (-NHNH2), succinimides (-ONC4O2), maleimide,thiols (-SH), halides (-X), silyl (-SiH), azido groups (-N3), phenyl groups^ sulfonates(SO3·), isothiocyanate, isocyanate, epoxides, nitrobenzyls, oxazoline, acid chloride,chloroformate, disulfide pyridyl, azlactone, cyanogen bromide, fluoroarenes,fluorocarbons, disulfides, isocyanides, sulfaamido, sulfate, heparin, peptides, nucleotides,polynucleotides, organic silicon compounds and organic phosphate (phosphoamidite)compounds.

For surfaces on which little or no specific surface binding is desired, inertfunctional groups 112 include non-reactive chemical groups such as, ethylene glycol 13 oligomers, acrylamides, pyrollidones, poly- and mono-saccharides and polar syntheticpolymers.

Choice of the functional group is within the skill of the art, based on knownbinding parameters of and derivatives of the functional group(s) appropriate for each use. 5 Thus, the active component can be designed to specifically or non-specifically select andassociate with chosen target analytes from external milieu.

The term "spacer," 114 as used herein, is intended to include those moieties thatserve to separate the functional group 110 or inert functional group 112 from the bindinggroup 116 of the active component 106. Typically, the length of the spacer group 114 is 10 sufficient when the functional group and binding group can perform their respective functions without physical or chemical interference from each other. The spacer links thefunctional group to the binding group and also preferably separates the functional group adistance away from the coated solid substrate 102. This spacer 114 increases theaccessibility of the functional group 110 to target species and reduces the steric hindrance 15 from or binding interference with the solid substrate 102.

It is preferable that the spacer 114 is one consistent length that provides all functional groups with the same activity; however, this is not required. Example spacersinclude, but are not limited to, hetero-, bi-functional or multi-functional small moleculesor polymers. These molecules can form covalent bonds or stable films upon solid 20 substrates and, at the same time, provide functional groups for specifically binding targetspecies from external milieu. For example, bi-functional, linear, star-shape, and comb-like PEGs (oligomers or polymers), polyethylenimines, polystyrene, polysiloxanes,polyurethanes, proteins, poly(amino acids), polyphosphazenes, telechelic blockcopolymers (Pluronics®), polyacrylates, polyacrylamides, polymethacrylates, 25 polysaccharides, dendrimers, hyperbranched polymers, macromonomers, and cross-linking or heterobifunctional alkyl-linked coupling reagents are suitable spacers.

The term "binding group," 116 as used herein, is intended to include thosemoieties that can bind the active component 106 to the coating matrix 100 and/or to theunderlying support 102. The binding group 116 is preferably selected to covalently bind 30 the active component to the coating matrix or to the substrate. The binding group canalso react with itself in the form of cross-linking. Examples of the binding group for usein the present invention include, but are not limited to, silanes, methacrylates, disulfides,disilazanes, sulfhydryls, acrylates, carboxylates, activated esters, other active leavinggroups, isonitriles, isocyanates, phosphoamidites, nitrenes, epoxides, hydrosilyl, esters, 14 arenes, azido, amine, nitrile, vinyl groups, alkylphosphonates, and known surface-coupling reactive species known to those skilled in the art of chemical coupling tosurfaces.

In a preferred embodiment, the active component 106 is produced by combining5 an amount of a reactive chemistry carrying the binding group portion, such as an amino- terminated alkyl silane, with a spacer bearing a terminal functional group, such as biotin-PEG-CO2-N-hydroxysuccinimidyl (NHS-PEG-biotin). This combination results in anactive component having a terminal silane available to act as the reactive or bindinggroup, a PEG portion acting as a spacer group, and biotin serving as the functional group. 10 The active component has the general molecular fonnula X-PEG-Y, where X is the functional group and Y is the binding group. Although a list of possible functionalgroups is provided above, example preferred functional groups include, but are notlimited to biotin, N-hydroxysuccinimide, vinylsulfone, metal ion chelates (e.g., Ni2+-NTA), glutathione binding group, amino, aldehyde, epoxy, mercapto, maleimide, heparin, 15 methoxy, and sulfonate. Example preferred binding groups include, but are not limited to silane, azide, acrylate, aldehyde, isocyanate, phosphonate, and epoxy.

The methods of the present invention preferably include forming a 0.001 mol/literto 0.1 mol/liter solution of active component 106 in a solvent, more preferably 0.005-0.02mol/liter solution of active component in a solvent, and most preferably 0.01 mol/liter 20 solution of active component in a solvent. As noted above, the active component 106 ispreferably a molecule of the form X-PEG-silane, wherein: X is a functional group such asbiotin, vinylsulfone, or an N-hydroxysuccinimidyl ester; PEG is a polyethyleneglycolpolymer, preferably of molecular weight 2000 to 5000 atomic mass units; and silane is ahydrolyzable silane precursor group (e.g., trialkoxy or trichlorosilane). 25 In a preferred embodiment, the active component 106 is a hiotin-PEG-silane molecule formed by reacting biotin-PEG-CO2-N-hydroxysuccinimide (biotin-PEG-NHS,molecular weight 3400, e.g., Shearwater Corp., OH2Z0F02) with an amino-terminatedalkoxysilane ((3-trimethoxysilylpropyl)-diethylenetriamine, e.g., Gelest, SIT8398.0) at a1:1 mole ratio in the chosen solvent. The reaction is preferably carried out at room 30 temperature for two hours. The N-hydroxysuccinimide group on the biotin-PEG-NHSreacts reliably with the terminal amine on the aminosilane to yield the biotin-PEG-silaneactive component. An example solvent for the active component is N,N- dimethylacetamide (DMAC, Aldrich 27,055-5). 15

<img img-format="tif" img-content="drawing" file="IL159556AD00024.tif" id="idf0004" />

In another preferred embodiment, two active components 106: biotin-PEG-silaneand methoxy-PEG-silane, are combined in the coating mixture. By diluting the biotinfunctional group in “inert” methoxy groups, increased biotin binding activity of boundstreptavidin can be achieved. The first active component is the biotin-PEG-silane, 5 molecular weight 3400 described in the previous paragraph. The second active component is a methoxy-PEG-silane, formed by reacting methoxy-PEG-OCLLCLL-CCVN-hydroxysuccinimide (methoxy-PEG-NHS), preferably of molecular weight less than3400, most preferably molecular weight 2000 (e.gv Shearwater Corp., 2M4M0D01), withan amino-terminated alkoxysilane ((3-trimethoxysilylpropyl)-diethylenetriamine, e.g., 10 Gelest, SIT8398.0) at a 1:1 mole ratio in the chosen solvent.

In another preferred embodiment, the active component is a methoxy-PEG-silane, preferably formed by reacting methoxy-PEG-OCH2CH2-CO2-N-hydroxysuccinimide,molecular weight 5000 (e.g., Shearwater Corp., 2M4M0H01) with an amino-terminatedalkoxysilane ((3-trimethoxysilylpropyl)-diethylenetriamine, e.g., Gelest, SIT8398.0) at a 15 1:1 mole ratio in the chosen solvent.

In another preferred embodiment, the active component 106 is a vinylsulfone- PEG-silane, preferably formed by reacting vinylsulfone-PEG-CO2-N-hydroxysuccinimide, molecular weight 3400 (e.g., Shearwater Corp. 2Z5B0F02) with anamino-terminated alkoxysilane ((3-trimethoxysilylpropyl)-diethylenetriamine, e.g., 20 Gelest, SIT8398.0) at a 1:1 mole ratio in the chosen solvent. , In another preferred embodiment 106, the active component is an NHS-PEG-silane, preferably formed by reacting a bis-functional N-hydroxysuccinimidyl ester ofPEG (SPA-PEG-SPA, molecular weight 3400, e.g. Shearwater Corp., 4M4M0F02) withan amino-terminated alkoxysilane ((3-trimethoxysilylpropyl)-diethylenetriamine, e.g., 25 Gelest, SIT8398.0) at a 2:1 mole ratio in the chosen solvent. In another preferred embodiment, the active component 106 is a COOH-PEG-silane, preferably fonned byreacting COOH-PEG-N-hydroxysuccinimide, molecular weight 3400 (e.g., ShearwaterCorp., OZ2ZOF12) with an amino-terminated alkoxysilane ((3-trimethoxysilylpropyl)-diethylenetriamine, e.g., Gelest, SIT8398.O) at a 1:1 mole ratio in the chosen solvent. 30 Cross-Linking Component and Matrix Forming Component For Use With The

Present Invention:

The “cross-linking component” 108 of the present invention is defined herein as areactive molecule with multiple reactive moieties that when activated by appropriate 16 stimulus, provides cross-linked stabilization within the film 100 as well as covalent oradsorptive film attachment to or upon various solid substrates 102.

In one embodiment, the cross-linking component 108 includes the alkylsilane andsulfonylazide reactive end groups, which when combined create dual reactive 5 functionalities that upon curing proceed to form spontaneous cross-links both within thecoating and to various substrates 102. Exemplary crosslinking groups include moleculesthat combine groups from the list including: methacrylates, acrylates, epoxides, silanes,perfluorophenyl azides, aryl azides, bis-nitrenes, acyl azides, azidoformates, sulfonylazides, phosphoryl azides, diazoalkanes, diazoketones, diazoacetates, beta-keto-alpha- 10 diazoacetates, aliphatic azo, diazirines, ketenes, photoactivated ketones, dialkylperoxidases, diacyl peroxidases, or quinones. As an example, 6- azidosulfonylhexyltriethoxysilane is provided, which includes silane groups able to formcovalent bonds with reactive hydroxyl groups present on surfaces of substrates such as aoxidized polymers, metal oxides, silicon wafers, silicates, titanates, aluminates, or 15 glasses. The silane groups can also cross-linked with each other or with other silanegroups, forming a three-dimensional cross-linked matrix. The azidosulfonyl group canform covalent bonds with aliphatic and aromatic compounds both on the substrate 102and/or within the coatable multicomponent matrix 104.

The tema "matrix-forming component," 104 as used herein, is intended to include 20 those molecules known in the art to have physical characteristics of providing low non-specific binding properties towards biological (e.g. proteins, polysaccharides, cells, andbacteria) and non-biological (e.g. particulate, colloidal, ionic, siliceous, andcarbonaceous) compounds. Ostuni, E.; Chapman, R. G.; Holmlin, R. E.; Takayama, S.;Whitesides, G. M. Langmuir, 17: 5605-5620 (2001). Such matrix-forming molecules 25 typically include one or more of the following characteristics: such molecules can bepolar, hydrophilic, and electrically neutral, are hydrogen bond acceptors, and typicallyexhibit conformational flexibility.

Examples of the matrix-forming component 104 include, but are not limited to,polyoxyethylene-based surface-active substances, including polyoxyethylene sorbitan 30 tetraoleate (PST), polyoxyethylene sorbitol hexaoleate (PSH), polyoxyethylene 6 tridecylether, polyoxyethylene 12 tridecyl ether, polyoxyethylene 18 tridecyl ether, Tweensurfactants, Triton® surfactants, and the polyoxyethlene-polyoxypropylene copolymerssuch as the Pluronic® and Poloxamer® product series (from BASE). Other matrix-forming components include dextrans, linear PEG molecules (MW 500 to 5,000,000), 17 star-shaped PEG molecules, comb-shaped and dendrimeric, hyperbrached PEGmolecules, as well as the analogous linear, star, and dendrimer polyamine polymers, andvarious carbonated, perfluorinated (e.g., DuPont Zonyl® fluorosurfactants) and siliconated(e.g, dimethylsiloxane-ethylene oxide block copolymers) surfactants. Matrix-forming 5 components 104 of biological origin include casein, serum dilutions, bovine serumalbumin, glycolipids and lipids, heparin and related glycosaminoglycans, muscin andpolysaccharides (dextrans, hyalurons, sepharose, cellulose, agarose, chondroitins,chitosans). A solution of cross-linking component 108 and matrix-forming component 104 is10 also formed in a carrier solvent. As noted above, the matrix-fonning component 104 ispreferably a linear or branched polyoxyethylene-containing surfactant. Most preferably, the matrix-fonning component 104 is polyoxyethylene sorbitan tetraoleate (PST, e.g.,Aldrich 46,639-5). The matrix-forming component is preferably dissolved to provide a0.01 to 5 % (vol/vol) solution in the final coating mixture. More preferably, the matrix- 15 forming component has a concentration of 0.5 to 2% in the final coating mixture. Mostpreferably, the matrix-forming component 104 concentration is 1% (vol/vol) in the finalcoating mixture.

As noted above, the cross-linking component 108 is preferably a bi-functional 1 chemically reactive compound, and more preferably a hetero bi-functional molecule with 20 azido functionality on one end of the molecule and alkyl silane functionality on the otherend. Most preferably, the cross-linking component 108 is an azidosilane (6-azidosulfonylhexyl-triethoxysilane, 95%; Gelest, e.g., SIA0780.0). The cross-linkingcomponent is preferably dissolved to give a 0.001 to 0.5 mol/liter solution, and morepreferably, 0.005 to 0.1 mol/liter solution in solvent. Most preferably, the cross-linking 25 component 108 is dissolved to provide a 0.02 mol/liter solution. The preferred solvent forthe cross-linking component/matrix-forming component mixture is dimethylsulfoxide(DMSO, e.g., Aldrich 47,230-1), although other solvents are envisioned.

Coating Solutions For Use In The Present Invention:

In use, the coating solutions 100 of the present invention are prepared by 30 combining an active component solution of the present invention with the cross-linkingcomponent 108/matrix-forming component 104 mixture of the present invention at avariety of different volume ratios. The most preferred ratio is approximately 1:4 volumeratio of active component solution to cross-linking/matrix-forming component solution.The most preferred final component concentrations in the coating solution are: 0.002 18 mol/liter active component (e.g., biotin-PEG-silane), 0.009 mol/liter matrix formingcomponent (polyoxyethylene sorbitan tetraoleate) and 0.0183 reactive component(azidosilane). '

Note that the most preferred final concentration depends in part on the coatingmethod to be employed and the desired coating thickness. The most preferredconcentrations described above are associated with a spin-coating fabrication operation.However, other active component to cross-linking/matrix-forming ratios and finalconcentrations are envisioned to be within the scope of the present invention as long asthe combinations provide useful coating solutions that exhibit the desired specific/non-specific binding characteristics. Note also that other target proteins or analytes maybeincluded in the combination of active component, cross-linking component, and matrix-forming component. For example, streptavidin may be mixed with biotinylated activecomponents to form a streptavidin-biotin surface, thereby providing a surface coatinghaving streptavidin immobilized within the coating matrix (termed a streptavidin-biotinsurface, see Example VIII below).

Substrates For Use In The Present Invention·.

The term "substrate," 102 as used herein, is art recognized and is intended toinclude any surface capable of being coated with the combination of components of thepresent invention. Suitable substrates 102 include refractive, transparent, adsorptive andopaque solid-phase materials. They include, but are. not limited to metals, such as, gold,platinum, silver, copper, iron, aluminum; polymers: such as polystyrene, polysulfone,polyetherimide, polyethersulfone, polysiloxane, polyester, polycarbonate, polyetber,polyacrylate, polymethacrylate, cellulose, nitrocellulose, perfluorinated polymers,polyurethane, polyethylene, polyamide, polyolefin, polypropylene, nylon, hydrogels, andrelated blends and copolymers; non-metals, such as silica, silicon dioxide, titaniumoxides, aluminum oxides, iron oxides, carbon, silicon, various silicates and other glasses,for example soda-lime glass, ceramics and sol-gels. Advantageously, the surfacechemistry of the present invention secures itself over all these classes of substrates 102and their derivatives and modifications. Again, it is believed that the cross-linking andother reactions within the matrix-forming component, reactive component and activecomponent facilitate the stability of the non-specific binding matrix upon and over thesubstrate 102 without unnecessary covalent attachment to the substrate.

Note that some substrates 102 for use in the present invention may requirecleaning steps and drying steps, oxidation steps or other procedures known in the art to 19 facilitate the effective uniform coating interaction between the coating solution 100 andthe target substrate 102. For example, glass slide substrates are typically “primed” byrinsing in high purity water, soaked in an alkaline glass cleaner, sonicated for a period ofapproximately 15 minutes in the cleaner, soaked in high purity water and finally blow dryunder N2 gas. Other substrate preparation procedures are discussed in the Examples orwould be known to one of skill in the art.

Application of The Coating Solution to a Substrate:

The coating mixture, as described above, is applied to a target substrate 102.Preferably the application of the coating solution to the substrate 102 is accomplished byspin coating, dip coating, spray coating, or any other type of coating or applicationmethod known to those of ordinary skill in the art. More preferably, the coating mixtureis spin coated onto the substrate. Most preferably, the coating mixture is spin coated ontothe substrate by applying 0.5 ml of the coating mixture to a substrate in a spin coater (e.g.,Laurell Model WS-200-4NPP/RPM/HSP-8K/VAC) and spinning at 3500 to 5000 rpm for90 seconds. Note that in certain applications a second, and sometimes third, coat orapplication of the coating mixture to the substrate is performed. These additional coatingapplications allow the thickness of the coating mixture layer to vary. Coating thicknesson a target substrate is typically from about 5 to 200 nm, and is preferably about 20 to 30nm. However, coatings can be of other thickness, as long as the coating is sufficient forperforming the functions of the herein described present invention.

Note that the combination of the above described components provide coatings100 that exhibit performance superior to the same coatings comprising subsets of thecomponents. As shown in Example XV below, embodiments of the present inventionoperate optimally when all the above-described components (active component 106,cross-linking agent 108, and matrix forming agent 104) are present in. the coating surface100. However, it is envisioned that embodiments of the present invention may excludeone of the above components and still maintain some level of function, albeit a function(specific and non-specific binding) at a potentially non-optimal manner.

Curing of Surface Coated Substrate Targets:

Once coated, curing, using known methods, stabilizes the coating onto thesubstrate. Preferably, the curing step is thermal activation in a vacuum oven. Morepreferably, the curing step includes a room temperature evacuation step in which thepressure within the vacuum oven is pumped down to a pressure of 150 mm Hg (absolute) 20 for a period of 30 minutes, after which the oven is heated to 40° to 140°C, most preferablyto 70°C. The total thermal curing step takes approximately one hour in this method. Thecuring temperature and time depend in part on the physical properties of the substratematerial. Hence, shorter or longer curing times may be required dependent on thetemperature and physical properties of the substrate 102. The coated surfaces are thenallowed to cool to room temperature. This multi-step curing process is beneficial becauseit provides solvent removal and sufficient reaction time and temperature to form a robust,adherent functional coating 100.

The curing step stabilizes the functional surface invention for use. Note that thecuring step can be by thermal activation (discussed above), photo activation, chemicalactivation or any other type of curing that creates cross-linking within the functionalsurface as well as bonding to the substrate. Thermal activation includes, for example,heating in an oven, photo activation includes, for example, irradiation with ultravioletlight, and chemical activation includes, for example, submersion in water, chemicallyfacilitating cross-linking Chemical cross-linking may also proceed without externalstimulus, for example through aging processes such as condensation. Any other form ofchemical and physical transformation or curing can be used as known by those ofordinary skill in the art.

Example Binding Assays For Use With The Present Invention:

In one embodiment of the present invention, a functional surface 100 forperformance of a biochemical or bioanalytical binding assay is provided. The functionalsurface comprises a general base composition tailorable to specific applications bychanging appropriate functional group chemistries within the coating base formulation ina cassette-style format readily adaptable to several distinct classes of interfacialproperties. The surface coating is engineered with the following components that permitfacile, customized, tailoring of the platform chemistry across a number of applications: anon-specific binding matrix affixed upon and/or to the substrate; and an active componentaffixed to the non-specific binding matrix.

The active component 106 is preferably the same as is described above withrespect to the method for producing a functional surface 100. The non-specific bindingmatrix facilitates adherence of the active component upon and/or to the substrate.Preferably, the non-specific binding matrix has a matrix-forming component and a cross-linking component. The matrix-forming component and the reactive component are 21 identical to those described above with respect to the method for forming a functional,selective-binding surface. Coating adhesion upon and/or to the underlying substrate isbelieved to be promoted by multiple non-covalent interactions (e.g., physicalentanglement, non-covalent binding) between the insoluble cross-linked film matrix and 5 substrate in addition to any covalent linkages directly to the substrate via chemicalreaction.

The surface chemistries of the present invention can be utilized in numerousanalytical systems, including immunodiagnostics, gene and protein microarrays andmicrofluidic “lab-on-a-chip” devices. Application of the surface chemistries to these 10 analytical settings are accomplished using the compositions and methods of the presentinvention in ways known to one of skill in the art. Example analytic assays for use withthe present invention include, but not limited to, TMB-microwell assay, which isdescribed in the following Examples, the staphylococcal enterotoxin B sandwich assay,also described in the following Examples, oligonucleotide microarray assays, also 15 described in the following Examples, as well as other like capture assays that quantify thepresence of antigens, pathogens, particles, drugs, toxins, and metabolites.

Note that different embodiments of the surface coating 100 of the presentinvention may be packaged together with a number of surface coating compatible, useful,target assay ingredients to provide kits appropriate for performing analytical assays. For 20 example, kits may include a solution of prepared surface coating, target substrate, forexample primed or un-primed glass slides, assay ingredients, for example, labeledantibody, and other helpful tools (pipettes, filters, tubes, etc). Note also, that kits may bepackaged with the substrate pre-coated with the target surface coating for use in theparticular assay of interest. 25 Further, the surface chemistries of the present invention can be utilized within the medical device implant field to limit or eliminate non-specific binding to the implanteddevice, examples include coatings to surfaces of existing devices such as catheters,shunts, pumps, filtration membranes, stents, valves, membranes and other devices. Suchcoatings are commonly used to reduce non-specific adsorption of biological components, 30 cells and microbes.

In another potential embodiment, the surface chemistries of the present inventioncan be utilized within the medical device implant field to provide coatings that provideactive functions. For example, the surface chemistries of the present invention can beused to entrap drugs for controlled release applications. In another example, the surface 22 chemistries of the present invention could he used to tether drugs, therapeutic proteins,antimicrobial compounds, etc. to the surface of a device.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided by way ofillustration and are not intended as limiting. EXAMPLE I:

Low Non-Specific Binding (NSB) Biotinylated Surface Coating Chemistryon a SiO2/Si Substrate

The following example illustrates the utility of an embodiment of the presentinvention for limiting NSB of a protein to a treated SiCty/Si substrate. A coating solutionwas prepared and used to form a low NSB, biotinylated, surface coating on a flat SiCty/Sisubstrate. Experiments tested the level of non-specific binding to either an untreatedSiCty/Si substrate or to the low NSB, biotinylated, surface-coated SiCty/Si substrate.

Coating Solution Preparation. An aminosilane solution in organic solvent wasprepared in a polypropylene vial by adding 26.5 μΐ (3-trimethoxysilylpropyl)-diethylenetriamine (Gelest) to 10 ml of solvent. Either dimethyl sulfoxide (DMSO) orη,η-dimethylacetamide (DMAC) can be used as the solvent (both from Aldrich). 1.0 mlof this solution was then added to a 40 mg aliquot of biotin-PEG-CCL-N-hydroxysuccinimidyl (Biotin-PEG-NHS, Shearwater Polymers, Inc.), where PEG is a3400 molecular weight polyethylene glycol. The NHS group of the Biotin-PEG-NHSreacts with the terminal amine on the amino silane to form a biotin-PEG-silane molecule.Tire biotin-PEG-silane/DMAC solution will be referred to as solution Ar

In a second vial, 70.6 μΐ of 6-azidosulfonylhexyl-triethoxysilane was added to 10ml DMSO. 125 μΐ of matrix forming agent (polyoxyethylene sorbitan tetraoleate, PST,Aldrich) was then added to this solution yielding a solution B. Solutions A and B werecombined in a 1:4 volume ratio (1 ml solution A added to 4 ml solution B) to give a finalcoating solution mixture used for spin coating onto target substrates.

Spin-Coating. 5-inch silicon wafers (p- or n-type, single-side polished, test gradewafers) were loaded in a spin coater (Laurell Technologies Corp.) and spun at 5000 rpm.The coating solution (0.5 ml) was dispensed onto the spinning wafer, and the wafer wasallowed to spin for 90 seconds.

Coated silicon wafers were placed in a vacuum oven pumped down to a vacuumof 150 mm Hg (absolute) for 30 minutes. The oven was then turned on and allowed to 23 heat to approximately 140°C. The total thermal treatment (heating ramp and hold) wasfor two hours. The wafers were then allowed to cool to room temperature in ambient air.

Six-millimeter diameter sample spots were defined on the wafer by stamping asilicone adhesive border pattern onto the surface. The wafer was then diced into square 5 pieces as is well known in the art and used in various assays.

Non-Specific Protein Binding (NSB) Assay. The following assay is illustrative of the non-specific binding levels of a model soluble globular serum protein on the coatingsolution treated wafer surface. An immunoglobulin G (IgG) antibody (160 kDa) wasused as the model non-specific binding protein. Specifically, a horseradish peroxidase 10 conjugate of rabbit anti-sheep IgG (KPL Laboratories) was dissolved in phosphate buffered saline (PBS) to provide a series of IgG concentrations, ranging from 0.1 to 10μg/ml. The sample to be assayed (“test piece”) was first triple-rinsed with high puritywater and blown dry with a stream of N2 gas. 20 μΐ of varying concentrations of the IgG-PBS solution were placed on test spots and incubated at 37°C in a 100% (nominal) 15 humidity chamber for 30 minutes. Various IgG test concentrations were used as described below. After incubation, the test spots were again triple rinsed with high puritywater and blown dry with N2 gas. 20 μΐ droplets of a commercial tetramethylbenzidine(TMB) peroxidase substrate were then added to each test spot. Each test piece wasincubated at 37°C in a 100% (nominal) humidity chamber for 30 minutes. 20 HRP catalyzes the formation of an insoluble, surface-deposited membrane in this assay, which can be detected colorimetrically or by ellipsometry. As such, the greater thelevel of NSB of horseradish peroxidase coupled IgG to the test piece, the greater the levelof surface-deposited membrane.

Control Experiments: Bare Substrates and BSA Blocking. In addition to the 25 coating solution treated substrates discussed above, two NSB control experiments werealso run in parallel·. (1) bare SiO2/Si wafer substrates ; and (2) SiO2/Si wafers blockedwith bovine serum albumin (BSA). BSA-blocking is currently the most widely usedtechnique for inhibiting non-specific protein binding on bioassay surfaces. Briefly, anuntreated piece of silicon wafer was triple-rinsed in high purity water and blown diy with 30 N2 gas. 40 μΐ droplets of a 1% BSA solution in PBS were then placed on each 6 mm testspot. The droplet covered the entire test spot, which was then incubated for 1 hour at37°C and 100% (nominal) relative humidity. The BSA solution was then rinsed off with 24 high purity water and blown dry with N2 gas, and the NSB assay described above wasperformed on the BSA-blocked SiO2/Si substrate.

The data in Fig. 2 illustrates that coated or treated SiO2/Si substrates of the presentExample showed little or no detectable IgG non-specific binding at any of the tested 5 concentrations (solid bars). In contrast, BSA-blocked wafers showed a much greater level of IgG NSB (open bars). Note the large error bars on the BSA-blocked surface data,indicating the IgG NSB variability commonly encountered with this blocking technique.NSB was very high on the bare SiO2/Si wafers, and increased off-scale for the 1 and 10pg/ml IgG protein loading (data not shown). 10 This data supports a conclusion that biotin functional surfaces of the present invention are effective at limiting or eliminating NSB of proteins to SiO2/Si substrates. Incontrast, bare SiO2/Si, and to a lesser extent BSA-blocked SiO2/Si substrates, showed agreater level of non-specific protein binding, illustrating again the effective utility of thepresent invention. 15 EXAMPLE Π:

Low Non-Specific Binding (NSB) Thiol-Reactive Surface Coating Chemistry on SiO2/Si Substrates

The following example illustrates the utility of another embodiment of the presentinvention for limiting NSB of a protein to a treated SiO2/Si substrate. A coating solution 20 was prepared and used to form a low NSB, thiol-reactive, surface coating on a SiO2/Sisubstrate. Experiments were performed to test the level of non-specific binding to eitheran untreated SiO2/Si substrate or to the low NSB, thiol-reactive, surface coated SiO2/Sisubstrate.

Coating Solution Preparation. The coating solution was prepared as essentially 25 described in Example I, with the exception that the Biotin-PEG-NHS was replaced withvinylsulfone-PEG-NHS (Shearwater). Spin coating was also performed as essentiallydescribed in Example I.

The spin coated wafers were placed in a vacuum oven pumped down to a vacuumof 150 mm Hg (absolute) for 30 minutes. The oven was then turned on and allowed to 30 heat to approximately 70°C. The total thermal treatment (heating ramp and hold) was forfour hours. The wafers were then allowed to cool to room temperature in ambient air. 25

Six-millimeter diameter sample spots were defined on the wafer by stamping asilicone adhesive border pattern onto the surface. The wafer was then diced and used invarious binding assays.

Non-Specific Protein Binding (NSB) Assay. Non-specific protein binding was5 assessed using a soluble TMB-microwell assay. Briefly, as described in Example 1, each test piece was rinsed with high purity water, dried, and incubated with a solution ofhorseradish peroxidase conjugate of rabbit anti-sheep IgG. Note that the PBS solution inthis Example included 0.1% Tween20 surfactant. After protein incubation and rinsing, 20μΐ droplets of a commercial, tetramethylbenzidine (TMB) soluble peroxidase substrate 10 solution were added to each test spot. 20 μΐ of stop solution was added to each TMBdroplet after 30 minutes incubation at 37°C and 100% (nominal) relative humidity. Thestop solution stops the enzymatic reaction and locks in the color change in the droplet.Droplets were combined using a micropipet and 80 μΐ samples were transferred from thetest piece to a microtiter plate. A plate reader monitoring optical density at 450 nm was 15 used to quantify the extent of the peroxidase reaction, winch is directly related to thequantity' of non-specifically bound IgG-HRP to each test piece.

Control Experiments: Bare Substrates and BSA Blocking, hi addition to thecoating solution treated substrates discussed above, two NSB control experiments werealso run in parallel: (1) bare SiO2/Si wafer substrates; and (2) SiO2/Si wafers blocked with 20 bovine serum albumin (BSA). As discussed in Example I, BSA-blocking is currently themost widely used technique for inhibiting non-specific protein binding on bioassaysurfaces. The BSA-blocking protocol was essentially as described in Example I. The testpiece was triple-rinsed in high purity water and blown dry with N2 gas. 40 μΐ droplets ofa 1% BSA solution in PBS were then placed on each 6 mm test spot. The droplet covered 25 the entire test spot, which was then incubated for 1 hour at room temperature and 100%(nominal) relative humidity. The BSA solution was then rinsed off with high purity waterand blown dry with N2 gas.

NSB Results: The data in Fig. 3 illustrates that coated or treated SiO2/Si substratesof the present Example showed little or no detectable IgG binding at any of the tested IgG 30 concentrations (solid bars). In contrast, BSA-blocked wafers showed a much greaterlevel of IgG NSB (cross-hatched bars). Note the large error bars on the BSA-blockedsurface data, indicating the IgG NSB variability commonly encountered with this 26 blocking technique. NSB was very high on the bare SiC^/Si wafers (open bars), andincreased off-scale for the ] and 10 pg/ml IgG protein loading (data not shown).

This data supports a conclusion that thiol-reactive functional surfaces of thepresent invention are effective at limiting or eliminating NSB of proteins to SiCVSisubstrates. In contrast, bare SiC^/Si, and to a lesser extent BSA-blocked SiC^/Sisubstrates, showed a greater level of non-specific protein binding, illustrating again theeffective utility of the present invention. EXAMPLE 111:

Low Non-Specific Binding (NSB) Biotinylated Surface Coating Chemistryon Plastic (Polymeric) Substrates

The following example illustrates the utility of an embodiment of the presentinvention for limiting NSB of a protein to a plastic (polymeric) substrate. A coatingsolution was prepared and used to form a low NSB, biotinylated, surface coating on theplastic substrate, e.g., polystyrene, polysulfone, polyetherimide, and polyethersulfone.Experiments were performed to test the level of non-specific binding to either anuntreated plastic substrate or to the low NSB, biotinylated, surface coated plasticsubstrate.

Polystyrene (PS). The polystyrene substrates consisted of 3-inch hydrophobicdisks approximately 2 mm thick. The disks were cut from bacteriological gradepolystyrene Petri dishes (VWR). The coating solution preparation was identical to thatdescribed in Example I. Prior to coating the polystyrene Petri dishes with the coatingsolution, an oxidation step was performed on the hydrophobic polystyrene surface.Oxidation of the surface was accomplished by submerging the disk in concentratedsulfuric acid (95% H2SO4, Mallinkrodt) for two minutes. The disks were then rinsedextensively with high purity water and blown dry with N2 gas. The disk was mounted inthe spin coater and spun at 5000 rpm. The coating solution (0.5 ml) was dispensed ontothe spinning disk and was allowed to spin for 90 seconds. At that point another 0.5 ml ofthe coating solution was dispensed and spun for 90 seconds. The PS surface thusreceived two coats of coating solution during the spinning process.

The coated PS disk was then placed in a vacuum oven that was evacuated to 150nun Hg (absolute) at room temperature for 30 minutes. After the 30-minute pump down,the oven was turned on and the samples were heated to 90°C under vacuum. The totaltime for thermal treatment (heat ramp and hold) was four hours. Each sample was then 27 allowed to cool to room temperature under ambient atmosphere. After cooling, 6 mm testspots were defined on the surface by stamping with a silicone adhesive border pattern.

Polysulfone (PSU), polyetherimide (PEI), andpolyethersulfone (PES). Thesesubstrates consisted of 2-inch square coupons cut from 0.02-inch thick commercial film 5 stock. The coupons were cleaned by sonicating for 15 minutes in a non-ionic detergent(Triton X-100) bath. After extensive high purity water rinsing, the coupons weresonicated for another 15 minutes in a 50:50 ethanol . water solution, followed by a waterrinse and N2 gas blow dry.

The coating solution preparation was essentially identical to that described in10 Example I. The coupons were mounted in the spin coater and spun at 5000 rpm. 0.3 ml of the coating solution was dispensed on the coupons and spun for 90 seconds. Thecoupons were then placed in a vacuum oven that was pumped down to a vacuum of 150mm Hg (absolute) for 30 minutes. The oven was then turned on and allowed to heat toapproximately 140°C. The total thermal treatment (heating ramp and hold) was for two 15 hours. The coupons were then allowed to cool to room temperature in ambient air. Six-millimeter diameter sample spots were defined on the wafer by stamping a silicone glueon the surface.

Non-Specific Binding (NSB) Assay. Non-specific protein binding was assessedusing a soluble TMB-microwell assay. First, coated PS, PSU, PEI or PES surfaces were 20 rinsed and incubated with the horseradish peroxidase conjugate of rabbit anti-sheep IgG(described in Example I), with the exception that the phosphate buffered saline alsocontained 0.1% Tween20 surfactant. After protein incubation and rinsing, 20 μΐ dropletsof a commercial, tetramethylbenzidine (TMB) soluble peroxidase substrate solution wasadded to each test spot. 20 μΐ of stop solution was added to each TMB droplet after 30 25 minutes incubation at 37°C and 100% (nominal) relative humidity. The stop solutionstops the enzymatic reaction and locks in the color change in the droplet. Droplets werecombined using a micropipet and 80 μΐ samples were transferred from the test piece to amicrotiter plate. A plate reader monitoring optical density at 450 nm was used to quantify . the extent of the peroxidase reaction, which is directly related to the quantity of non- 30 specifically bound IgG-HRP to each surface. NSB Results. Fig. 4 provides representative non-specific· protein (IgG) binding results. The data presented compare the performance of our surface coating (solid bars)relative to traditional bovine serum albumin (BSA) blocking (open bars). The data 28 159,556/2 correspond to incubation in 10 pg/ml IgG for each of the plastic substrate (PS, PSU, PEI,PES), and show that our surface coating is more effective in inhibiting NSB than BSA-hlocking on these widely used plastic substrates. EXAMPLE IV:

Low Non-Specific Binding (NSB) Biotinylated Surface Coating Chemistryon Metal/Metal Oxide Substrates

The following example illustrates the utility of an embodiment of the presentinvention for limiting NSB of a protein to a metal/metal oxide substrate. A coatingsolution was prepared and used to form a low NSB, biotinylated, surface coating on themetal/metal oxide substrate. Experiments were performed to test the level of non-specificbinding to either a BSA blocked metal/metal oxide substrate or to the low NSB,biotinylated, surface coated metal/metal oxide substrate.

The metal substrate consisted of a 30 nm gold film vapor deposited onto a 5 -inchsilicon wafer. A ~5 nm chromium adhesion interlayer was used between the gold andsilicon oxide. The coating solution preparation was identical to that described in Example I. The gold-coated wafer was mounted in the spin coater and spun at 5000 rpm. 0.5 ml ofthe coating solution was dispensed onto the wafer that was spun for 90 seconds. Asecond application of 0.5 ml coating solution was dispensed onto wafer and spun foranother 90 seconds. As such, the metal/metal oxide substrate surface received two coatsof coating solution during the spinning process.

The coated wafer was then placed in a vacuum oven that was pumped down to avacuum of 150 mm Hg (absolute) for 30 minutes. The oven was then turned on andallowed to heat to approximately 140°C. The total thermal treatment (heating ramp andhold) was for two hours. The wafers were then allowed to cool to room temperature inambient air. Six-millimeter diameter sample spots were defined on the wafer by stampingsilicone adhesive border pattern on the surface. Non-specific binding of protein wasassessed using the soluble colorimetric TMB method as described in Example Π. NSB Results: Fig. 5 provides representative non-specific protein (IgG) bindingresults. The data presented compare the performance of our surface coating relative totraditional bovine serum albumin (BSA) blocking. The data shows that the coated surface(solid bars) is superior to the traditional BSA-blocking approach (open bars) at an IgGprotein solution load of 10 μβ/ιηΐ. At lower protein concentrations, both surfaces areeffective at inhibiting NSB. 29

This data supports a conclusion that biotinylated reactive functional surfaces ofthe present invention are effective at limiting or eliminating NSB of proteins tometal/metal oxide substrates. In contrast, BSA-blocked metal/metal oxide substrates, at10 pg/ml or higher concentration, showed a greater level of non-specific protein binding,illustrating again the effective utility of the present invention. EXAMPLE V:

Low Non-Specific Binding (NSB) Thiol-Reactive Surface Coating Chemistryon Glass Microscope Slides

The following example illustrates the utility of an embodiment of the presentinvention for limiting NSB of a protein to a glass microscope slide. A coating solutionwas prepared and used to form a low NSB, thiol-reactive, surface coating on the glassslide substrate. Experiments were performed to test the level of non-specific binding toeither a bare glass slide, a BSA blocked glass slide or to the low NSB, thiol-reactive,surface coated glass slide.

Substrates (glass slides) used in this particular example were polished 25 x 75 mmmicroscope slides obtained from a commercial vendor (TeleChem SuperClean™) andused as received. Note, however, that several glass microscope slide substrates have beensuccessfully used, including plain soda-lime glass slides.

Prior to coating, the slides were cleaned by the following protocol. First the slideswere rinsed with high purity (IS ΜΩ-cm) water. Slides were then loaded in a glassstaining rack, submerged in a 60°C, 1% Alconox solution (alkaline glass cleaner) andsonicated for 15 minutes. The slides were then rinsed with copious amounts of highpurity water and sonicated in high purity water for another 15 minutes. Slides were thenallowed to soak in fresh ultrapure water. Finally they were blown dry with N2 gas. Theslides were coated within two hours of cleaning.

Pre-primed glass slides have also been successfully coated. Primer coats includevapor deposited S1O2 layers and spin-on glass formulations such as Seramic™ (Gelest).

The coating solution was prepared as described in Example Π. Spin coating wasperformed as follows: a glass slide was mounted on the vacuum chuck of the spin coater.0.5 ml of coating solution was dispensed on the stationary slide. The spinner was thenturned on and accelerated to 3500 ipm, where it spun for 90 seconds.

The vacuum and thermal cure processes were identical to those described inExample H. The NSB assay and control experiments were performed as described in 30

Example IV, with the following changes. After the 5 minutes TMB peroxidase substrateincubation, 20 μΐ of TMB-Stop solution was added to the test spots to stop thecolorimetric reaction. Optical density was then read directly with the glass slide mountedon a 96-well plate holder (there was no fluid transfer to a microtitre plate).

Binding Results: Results are presented in Fig. 6. These results show that thecoated glass surface (solid bars) shows significantly lower non-specific protein bindingthan BSA-bloCked (cross-hatched bars) and bare glass substrates (open bars).

This data supports a conclusion that thiol-reactive functional surfaces of thepresent invention are effective at limiting or eliminating NSB of proteins to a glass slidesubstrate. In contrast, bare glass sides and BSA-blocked glass slides showed a greaterlevel of non-specific protein binding, illustrating again the effective utility of the presentinvention. EXAMPLE VI:

Low Non-Specific Binding (NSB) of Fibrinogen to Coated Surface

The following example illustrates the utility of an embodiment of the presentinvention for limiting NSB of fibrinogen to coated surfaces of the present invention. Acoating solution was prepared and used to form a low NSB, thiol-reactive, surface coatingon a SiC^/Si substrate. Experiments were performed to test the level of non-specificbinding of fibrinogen to either a bare substrate, a BSA blocked substrate or to the lowNSB, thiol-reactive, surface coated substrate.

Coated surfaces were prepared as described in Example II.

Fibrinogen Non-Specific Protein Binding Assay. Non-specific fibrinogen bindingwas assessed using a soluble TMB-microwell assay as follows: test pieces were rinsedand test wells were incubated for 30 minutes with serial dilutions of human fibrinogen(Sigma) in phosphate buffered saline (PBS). After protein incubation, the substrates wererinsed three times with PBST (PBS with 0.01% Tween20) and one time with clean waterand then blown dry. Each test well was then incubated for 30 minutes with a 20 μΐdroplet of a 1:8000 dilution of peroxidase labeled anti-human fibrinogen (Abeam) inPBST. The slides were rinsed as above and then 20 μΐ droplets of a commercial,tetramethylbenzidine (TMB) soluble peroxidase substrate solution. The TMB assay andoptical density measurements were performed as described in Example IV. 31

<img img-format="tif" img-content="drawing" file="IL159556AD00025.tif" id="idf0005" />

Control Experiments: Bare Substrates and BSA Blocking. In addition to thecoated substrates, two NSB control experiments were run in parallel, as essentiallydescribed in Example II. NSB Results: Non-specific binding results are presented in Fig. 7. The coated5 surfaces (solid bars) show very low non-specific fibrinogen binding relative to the bare (open bars) and BSA-blocked (cross-hatched bars) surfaces.

This data supports a conclusion that thiol-reactive functional surfaces of thepresent invention are effective at limiting or eliminating NSB of fibrinogen to a targetsubstrate. In contrast, non-treated substrate and BSA-blocked substrate showed a greater 10 level of non-specific fibrinogen binding, illustrating again the effective utility of the present invention, especially in light of fibrinogens relative abundance as a serum protein. EXAMPLE VII:

Demonstration of Selective versus Non-Specific Protein Binding on CoatedSurfaces 15 The following example illustrates the utility of an embodiment of the present invention for demonstrating selective protein (streptavidin) binding versus non-specificprotein binding on biotinylated coatings. The test pieces in this study were film-coatedsilicon wafers, wherein the biotinylated coating was prepared as described in Example I.Control test pieces were also prepared as previously described except that control surfaces 20 were prepared with non-reactive methoxy-capped PEG molecules instead of biotin.

Specifically, these coatings were formed by replacing the biotin-PEG-NHS molecule inExample I with a methoxy-PEG-succinimidyl propionate (mPEG-SPA, MW 5000).Otherwise, all processing steps were identical to those in Example I.

Streptavidin binding to these surfaces was assessed using horseradish peroxidase 25 conjugated streptavidin (SA-HRP, Pierce) and the soluble TMB-Microwell assay described in Example H. Various concentrations of SA-HRP were prepared in phosphatebuffered saline with 0.01% Tween20 surfactant. 20 μΐ drops of SA-HRP solution wereincubated on test spots for 30 minutes at 37°C and 100% (nominal) relative humidity.

The test spots were then rinsed with high purity water. The soluble TMB-microwell 30 assay described in Example II was used to assess binding.

Binding Results: Fig. 8 shows representative results. These results show that the biotinylated functional surface (open bars) binds significantly more streptavidin than themethoxy-PEG control surface (“inert” surface) (solid bars). Given the previous non- 32 specific protein binding assay results (see Example I), we conclude that the SA isspecifically bound to the biotinylated coating. The small signal observed on the methoxy-capped PEG sample for a SA load of 10 ug/ml is likely, the result of a small amount ofnon-specific streptavidin binding. 5 This data supports a conclusion that functional surfaces of the present invention are effective at providing selective binding sites on a coated substrate, while inertfunctional surfaces of the present invention are effective at limiting non-specific bindingon a coated surface. EXAMPLE VIII: 10 Demonstration of Biotinylated Molecule Binding to Streptavidin-Biotin CoatedSurfaces

The following example illustrates the utility of an embodiment of the presentinvention for demonstrating the fabrication of a streptavidin-biotin surface and thesubsequent selective binding of a biotinylated molecules to this surface. 15 Coating Solution Preparation. A first aminosilane solution was prepared in a polypropylene vial by adding 26.5 μΐ (3-trimethoxysilylpropyl)-diethylenetriamine(Gelest) to 10 ml of Ν,Ν-dimethylacetamide (DMAC). 1.0 ml of this solution was thenadded to a 40 mg aliquot of biotin-PEG-CO2-N-hydroxysuccinimidyl ester (Biotin-PEG-NHS, Shearwater Corp.), where PEG is a 3400 molecular weight polyethylene glycol. 20 The NHS group reacts with the terminal amine on the amino silane to form a biotin-PEG-silane molecule. The biotin-PEG-silane/DMAC solution is called solution A. In a secondvial, 70.6 μΐ of 6-azidosulfonylhexyl-triethoxysilane was added to 10 nd DMSO. 125 μΐof matrix forming agent (polyoxyethylene sorbitan tetraoleate, PST, Aldrich) was thenadded to this solution, which is called solution B. Solution A and B were then combined 25 in a 1:4 volume ratio (1 ml solution A added to 4 ml solution B) to give a final mixturecalled the biotin-PEG solution. A second aminosilane solution was prepared in a polypropylene vial by adding48.4 μΐ (3-trimethoxysilylpropyl)-diethylenetriamine (Gelest) to 10 ml of Ν,Ν-dimethylacetamide (DMAC). 1.0 ml of this solution was then added to a 40 mg aliquot of 30 methoxy-PEG-succinimidyl propionate (mPEG-SPA, Shearwater Corp.), where PEG is a2000 molecular weight polyethylene glycol. The NHS group reacts with the terminalamine on the amino silane to form a methoxy-PEG-silane molecule. The methoxy-PEG-silane/DMAC solution is called solution C. Solution C and B (described above) were 33 then combined in a 1:4 volume ratio (1 ml solution A added to 4 ml solution B) to give afinal mixture called the methoxy-PEG solution.

The biotin-PEG solution was then combined with the methoxy-PEG solution in a1:4 volume ratio. This final mixture is used to coat a silicon substrate. 5 Spin-Coating. Spin-coating and the thermal cure were performed as described in

Example I. Six-millimeter diameter sample spots were defined on the wafer by stampinga silicone adhesive border pattern onto the surface. The wafer was then diced and used invarious assays.

Formation of the Streptavidin Layer. Streptavidin (Prozyme, Inc.) was diluted to10 100 pg/ml in phosphate buffered saline (PBS). 40 μΐ droplets were then incubated in sample spots for one hour at room temperate. Several spots on the wafer were incubatedwith PBS only. These are the “no streptavidin” control spots. After incubation, thewafers were rinse three times with PBS-Tween20 (PBST) and one time with ultrapurewater and were then blown dry. This process results in a streptavidin immobilized within 15 the coating matrix.

Specific and Non-Specific Binding Assays. Biotinylated horseradish peroxidase(biotin-HRP, Pierce) was diluted to 10 pg/ml in PBST. 20 μΐ droplets were thenincubated on test spots for 30 minutes at room temperature. Peroxidase labeled rabbitanti-sheep IgG (IgG-HRP) was used as a non-biotinylated control molecule. The IgG- 20 HRP was diluted to 10 pg/ml in PBS (no Tween20), and 20 μΐ droplets were incubated ontest spots for 30 minutes at room temperature.

Results are presented in Fig. 9. The streptavidin-biotin surface showssignificantly higher signal for biotin-HRP (solid bars) than for the non-biotinylated IgG-HRP (open bars). Relative to the streptavidin surface, neither biotin-HRP nor IgG-HRP 25 show significant binding to the no-streptavidin control. The slightly higher signal for theIgG-HRP on the no-streptavidin surface can be attributed to the different incubationbuffers. In the absence of Tween20 in the buffer, the IgG-HRP shows slightly highernon-specific binding.

This data supports a conclusion that biotinylated molecules specifically bind to 30 streptavidin-biotin surfaces of the present invention, and conversely, that non-specific binding proteins show limited binding to these same coated surfaces. This Example againhighlights the utility of the present invention. 34 % EXAMPLE IX:

Demonstration of Thiolated Protein Attachment to Thiol-ReactiveCoatings on Polymer Substrates

The following example illustrates the utility of an embodiment of the present5 invention for selectively attaching a thiolated protein on a thiol-reactive coating chemistry on a plastic substrate. Results are compared to binding of the thiolated protein to an inertcontrol surface chemistry on a plastic substrate.

Coating Preparation. The coating solution was prepared as described in ExampleI, with the exception that the Biotin-PEG-NHS was replaced with vinylsulfone-PEG-NHS 10 (Shearwater). Spin coating on tissue culture polystyrene (TCPS) was performed asdescribed in Example III.

The coated wafer was then placed in a vacuum oven and was subsequentlypumped down to a vacuum of 150 mm Hg (absolute) for 30 minutes. The oven was thenturned on and allowed to heat to approximately 70°C. The total thennal treatment 15 (heating ramp and hold) was four hours. The substrates were then allowed to cool to room temperature in ambient air. Six-millimeter diameter sample spots were defined onthe surfaces by stamping a silicone adhesive border pattern onto the surface.

Inert Coating Preparation. The inert control surfaces were prepared exactly asdescribed in Example VH. 20 The specific binding assays were performed at room temperature as follows: surfaces were first triple-rinsed with ultrapure (18 ΜΩ-cm) H2O and then blown dry withN2 gas. Each test spot was then incubated with a 20 μΐ droplet of thiolated streptavidin inborate buffer (50 mM borate, 1 mM EDTA, pH 9.0, 0.01% Tween20). Incubation was atroom temperature for 30 minutes. Note that no blocking step was used. After incubation, 25 surfaces were triple-rinsed with PBST (0.1 M sodium phosphate, 0.15 M sodium chloride,pH 7.2, 0.01 % Tween20) and blown dry. Test spots were incubated with a 1:100 dilutionof biotin horseradish peroxidase (Pierce) for 30 minutes, rinsed with PBST, and thenblown dry. Test spots were then incubated with a 20 μΐ droplet of TMB substrate. Theenzyme catalyzed colorimetric TMB reaction was allowed to proceed for 5 minutes at 30 which point it was stopped by applying a 20 μΐ droplet of stop solution. Optical densityat 450 nm was read using a standard plate reader.

Binding Results: Specific binding results for the coated tissue culture polystyrenesubstrate are presented in Fig. 10 (note that each concentration was run in triplicate and 35 error bars represent one standard deviation in the set). Binding of thiolated streptavidin tothiol-reactive coatings has been demonstrated (open bars). The dose response on thethiol-reactive coatings scales with-concentration of thiolated streptavidin. Peroxidaseactivity on the inert surface is minimal (solid bars). The lack of non-specific binding on 5 the inert surfaces provides solid evidence of covalent attachment of streptavidin to thethiol-reactive coatings. EXAMPLE X:

Demonstration of Protein Attachment to Amine-Reactive Coatings onGlass Microscope Slides 10 The following example illustrates the utility of an embodiment of the present invention for selectively attaching an amine-containing protein to the amine-reactivecoating chemistry, compared to a deactivated control surface, on glass microscope slides.

Coating Preparation. The coating solution was prepared as described in ExampleI, with the exception that the Biotin-PEG-NHS was replaced with a 80 mg aliquot of 15 SPA-PEG-SPA (Shearwater) where SPA is a succinimidyl derivative of propionic acidthat exhibits reactivity towards amine groups. Glass substrates in a 25 x 75 mmmicroscope slide format were primed with an approximate 400 angstrom RF sputteredsilicon oxide layer and then cleaned by the following protocol. The slides were firstrinsed with high purity (18 MW-cm) water to remove gross impurities. They were then 20 loaded in a glass staining rack, submerged in an outgassed solution of 1 % Alconox solution heated to 60°C (alkaline glass cleaner) and sonicated for 15 minutes. The slideswere then rinsed with copious amounts of high purity water and then sonicated in highpurity water heated to 60°C for another 15 minutes. The slides were then rinsed withcopious amounts of high purity water and then placed in fresh high purity water until the 25 drying step. Slides were exhaustively blown dry with compressed N2 gas and were storeddry until use. The cleaned and primed slides were mounted in the spin coater and spun at3500 rpm. 0.5 ml of the coating solution was dispensed onto the primed glass slide andspun for 90 seconds.

The coated 25 x 75 mm glass slide was then placed in a vacuum oven and was 30 subsequently pumped down to a vacuum of 150 mm Hg (absolute) for 30 minutes. Theoven was then turned on and allowed to heat to approximately 70°C. The total thermaltreatment (heating ramp and hold) was 1 hour. The substrates were then allowed to cool 36 to room temperature in ambient air. Six-millimeter diameter sample spots were definedon the surfaces by stamping a silicone adhesive border pattern onto the surface.

Specific Binding Assay: The specific binding assays were performed at roomtemperature as follows. Surfaces were first triple-rinsed with ultrapure (18 ΜΩ-cm) H2O 5 and then blown dry with N2 gas. An inert control surface was generated by chemicallydeactivating an amine-reactive coating using a 50 mM ethanolamine in a 50 mM boratebuffer, pH 9.0 for 1 hour. Each test spot was then incubated with a 20 μΐ droplet ofstreptavidin in 50 mM phosphate buffer at pH 7.0. Incubation was at room temperaturefor 60 minutes. No blocking step was used. After incubation, surfaces were triple-rinsed 10 with PBST (0.1 M sodium phosphate, 0.15 M sodium chloride, pH 7.2, 0.01% Tween20)and blown dry. Test spots were incubated with a 1:100 dilution of biotin horseradishperoxidase (Pierce) for 30 minutes, rinsed with PBS (0.1 M sodium phosphate, 0.15 Msodium chloride, pH 7.2), and then blown dry. Test spots were then incubated with a 20μΐ droplet of TMB substrate. The enzyme catalyzed colorimetric TMB reaction was 15 allowed to proceed for 5 minutes at which point it was stopped by applying a 20 μΐ droplet of stop solution. Optical density at 450 nm was read using a standard plate reader.

Binding Results: Specific binding results for the amine-reactive coating are presentedin Fig. 11. Streptavidin immobilization to the amine-reactive coatings has beendemonstrated (open bars). The dose response on the amine-reactive coatings scales with 20 concentration of streptavidin. Peroxidase activity on the deactivated surface is minimal(solid bars). The lack of non-specific binding on the deactivated coatings provides solidevidence for the covalent attachment of streptavidin to the amine-reactive coatings. EXAMPLE XI:

Demonstration of Selective binding of biotinylated antibody to 25 streptavidin Coated Surfaces

The following example illustrates the utility of an embodiment of the present invention for demonstrating the selective binding of a biotinylated antibody to a coatedsurface of the present invention.

Surface Fabrication. Glass microscope slides with amine reactive surface 30 chemistries were prepared as described in Example X. Six-millimeter diameter samplespots were defined on the wafer by stamping a silicone adhesive border pattern onto thesurface. Streptavidin was covalently attached through its surface amine using thefollowing procedure. Streptavidin (Prozyme, Inc.) was diluted to 100 pg/ml in phosphate 37 buffer (50 mM sodium phosphate, pH 7.0). 40 μΐ droplets of this solution were thenincubated in sample spots for one hour at room temperate. The slides were then rinsedand the submerged in a deactivation solution consisting of 50 mM ethanolamine in 50mM phosphate buffer, pH 7. The deactivation was allowed to proceed for 1 hour, atwhich point the slides were removed from the solution, rinsed with water and dried.

Assay. A biotinylated goat antibody (goat anti-mouse IgG, biotin-labeled, Pierce)was used as the positive sample. The control was a non-biotinylated goat antibody (goatanti-human myoglobin, ICN). Both antibodies were diluted to 100 μβ/τηΐ in phosphatebuffered saline containing 0.005% Tween20 (PBST). 20 μΐ droplets of each antibodysolution were incubated in sample spots for 30 minutes at room temperate. Severalcontrol spots were incubated with PBST only (no antibody). Slides were then rinse threetimes with PBST, one time with ultrapure water, and then dried. Goat antibodyimmobilized on the surface was assessed using immunochemistry. Specifically, 20 μΐdroplets of a peroxidase labeled anti-goat antibody (rabbit anti-goat IgG-HRP, KPL Inc., 1:100 dilution in PBST) were incubated on all test spots for 30 minutes at roomtemperature. The slides were then rinsed as above and incubated with 20 μΐ droplets ofperoxidase substrate (TMB, KPL Inc.) for 5 minutes, at which point the colorimetricreaction was stopped with a TMB stop solution. Droplets were transferred to a 96-wellmicrotitre plate and optical density at 450 nm was measured using a plate reader (OpsysMR, Thermo Labsystems).

Binding Results: Results are presented in Fig. 12. The data illustrates that in theabsence of the first layer capture antibody (capture antibody concentration - 0), the non-specific binding of the detection antibody (anti-goat, HRP) is very low (open bars). Onsurfaces incubated with the goat antibodies, results show that only the biotinylatedantibody gives significant signal (solid bars). These results demonstrate that the surfaceembodiments of the present invention are selective for biotinylated antibody. EXAMPLE XII:

Demonstration of Improved Immunoassay Performance on Coated Surfaces

The following example illustrates the utility of an embodiment of the presentinvention by comparing the assay performance and detection limits of the coatingchemistries of the present invention relative to a standard diagnostic assay. Thecomparison is based on sandwich enzyme-linked immunosorbent assay (ELISA) forstaphylococcal enterotoxin B (SEB). 38 * SEB Sandwich Assay Protocol for Coated Substrates. The substrates for thisassay consisted of SiO^/Si wafers coated with biotinylated, low-NSB surface coating asdescribed in Example I. Streptavidin was bound to the surface by incubating 25 mg/ml(streptavidin in PBST) droplets (20 μΐ) at each 6 mm test spot. Incubation was for 30

5 minutes at 37°C. Each test piece was then triple-rinsed with high purity (18 ΜΩ-cm)water and blown dry with N2 gas. Capture antibody was bound to each test spot byincubating with 20 μΐ droplets of biotin anti-SEB (Toxin Technologies). Incubation wasperformed for 30 minutes at 37°C followed by a triple rinse in high purity water and theN2 gas drying step. Test spots were then incubated with 20 μΐ droplets of the SEB 10 antigen solution in concentrations ranging from 0.01 ng/ml to 1.0 ng/ml. Incubation wasagain performed for 30 minutes at 37°C. Test pieces were then triple rinsed with highpurity water and blown dry. Detection antibody (anti SEB-EERP in PBST, 20 μΐ droplets,Toxin Technologies) was then incubated for 30 minutes at 37°C, followed by a triplerinsing with high purity water and blow dry. The final incubation was with a commercial, 15 precipitate TMB assay. TMB-Membrane substrate (KPL, Inc.) was added in 20 μΐdroplets to each of the test spots and incubated for 30 minutes at 37°C. As describedpreviously, HRP catalyzes the formation of an insoluble deposit on the surface. Afterblowing the test piece dry with N2, the thickness of the deposited layer was quantifiedusing a custom-built ellipsometer, providing an indirect, quantitative measurement of 20 antigen at the surface. SEB Sandwich ELISA Protocol. A standard ELISA format assay was performedfor comparison to the SEB sandwich assay for coated surfaces discussed above. Briefly,100 μΐ of capture antibody (biotin anti-SEB, Toxin Technology) in PBS-Tween (0.01%)was added to the wells of a 96-well microtiter plate (Dynex Technologies, Immulon 2HB) 25 and was incubated for 120 minutes at 37°C. The wells were then triple rinsed with PBSsolution, tapping fluid from the plate after each rinse. The wells were then blocked withbovine serum albumin (BSA) by adding 200 μΐ of 1% BSA in PBST and then incubatingat 37°C for 1 hour. SEB antigen solutions (Toxin Technology, 100 μΐ volumes withconcentrations ranging from 0.01 ng/ml to 1.0 ng/ml in PBST) were added to the

30 micro wells and incubated at 37° for 60 minutes. The wells were then rinsed with PBST and fluid was tapped from the inverted plate. The detection antibody was a horseradishperoxidase conjugated anti-SEB (Toxin Technology). 100 ml volumes were incubated ineach of the wells for 60 minutes at 37°C. Again, the wells were washed with PBST. 39

Color was developed in the well by adding a commercial TMB peroxidase substrate(TMB-Microwell, KPL, Inc.). 100 μΐ volumes of the developer were added to the wellsand were incubated for 60 minutes at 37°C. After 60 minutes, 100 ml of the TMB stopsolution was added to each well, and the optical density at 450 nm was read using a 5 microplate reader.

Binding Results. Representative assay results are presented in Fig. 13A, results ofthe standard microtiter plate ELISA assay, and Fig. 13B, results for the ELISA assayperformed on the functional coated surface of this invention. The important observationis that the coated surface, which presents specifically bound streptavidin for the sandwich 10 assay, enables an order of magnitude improvement in assay sensitivity relative to a traditional sandwich ELISA. Specifically, the lower detection limit in the microtiter plateELISA was approximately 1.0 ng/ml SEB. On the coated surface of the presentinvention, the lower detection limit was 0.1 ng/ml SEB. EXAMPLE XIII: 15 Surface Chemistry Comparison in Oligonucleotide Microarray FormatThe following example illustrates the utility of an embodiment of the present invention as coating chemistries for use in oligonucleotide microassay formats. Theexperiments described in this example evaluate one coating chemistry embodiment of thepresent invention in parallel with current “state-of-the-art” commercial polymer and 20 silane chemistries. In particular, the signal, noise and signal-to-noise levels are comparedfor the chemistries in a microarray format using amine labeled oligonucleotides.

Coating Preparation: The coating chemistry was prepared exactly as described inExample X. The current state of the art commercial polymer chemistry is advertised as athree dimensional polymer coating on a glass slide substrate with a high loading capacity 25 for amine labeled oligonucleotide. The commercial silane chemistry is advertised as alow background silane coating on a glass slide substrate. The commercial chemistrieswere processed according to the manufacturer suggested protocols, which are known anduniform for these types of substrates (two such manufacturers include:

Microarray Printing: 5’ amine-linked and 3’ biotin-labeled oligonucleotide 30 (Operon) was suspended in 300mM sodium phosphate buffer, pH 8.5 at a concentrationof 20 μΜ. The coating chemistry was first triple-rinsed with ultrapure (18 ΜΩ-cm) H2Oand then blown dry with N2 gas prior to arraying. Commercial chemistries were used outof the box. An array of spots was printed on the chemistries using a SpotBot 40 microarrayer (Telechem) fitted with SMP3B pins (Telechem). Commercial polymer andcoating chemistries were incubated at 75% relative humidity for 5 hours. The polymer,silane and coating chemistries were stored desiccated at room temperature for 72 hoursprior to use. The coating chemistry was deactivated with 50mM ethanolamine inphosphate buffer pH 7.0 for 1 hour.

Specific Binding Assay: The commercial polymer chemistry was deactivated witha 50mM ethanolamine in 0.1M Tris, pH 9.0, 1%SDS solution at 50° C for 20 minutes.

The commercial silane chemistry was baked at 80°C for 2 hours then blocked with bovineserum albumin for 1 hour. The specific binding assays were performed at roomtemperature as follows: chemistries were incubated with 140 μΐ of 10 /ig/ml of Cy5labeled streptavidin (Amersham) in PBST (0.1 M sodium phosphate, 0.15 M sodiumchloride, pH 7.2, 0.01% Tween20). 40 mm Lifter Slips (Erie) were utilized to spreadliquid evenly over surface of slide chemistries. Chemistries were placed in hybridizationchamber for 30 minutes. Lifter Slips were removed from slide chemistries and slideswere triple-rinsed with PBST then ultrapure (18 ΜΩ-cm) H2O and dried. Slidechemistries were placed in GenePix 4000B scanner (Axon) and settings were optimizedfor the commercial polymer slide chemistry. Final scanner settings were 100% laserpower and 500 volt setting on the photo multiplier tube.

Binding Results: Specific binding results for the coating chemistry andcommercial chemistries are presented in Fig. 14A. Signal intensities are reported inrelative fluorescence units. Fig. 14A shows that the coating chemistry (open bar) hashigher signal intensity than both the polymer (cross-hatched bar) and silane (solid bar)chemistries in this experiment.

The background was monitored in the Cy5 channel. Noise levels are reported inrelative fluorescence units. Representative non-spotted areas of the scanned image wereused to calculate global background values. The Cy5 channel signal provides ameasurement of the non-specific binding contribution to background signal. Fig. 14Bshows the coating chemistry (open bar) has significantly lower Cy5 backgroundcompared to polymer (cross-hatched bar) and silane (solid bar) chemistries. The coatingchemistry has lower background signal due to low surface non-specific binding of theCy5 streptavidin.

Resulting signal-to-noise ratios in the Cy5 channel were calculated for the slidechemistries and are shown in Fig. 14C. The coating chemistry (open bar) shows 41 approximately 3x higher signal-to-noise ratio than the commercial polymer chemistry(cross-hatched bar) and a 6x improvement in signal-to-noise over the commercial silanechemistry (solid bar). EXAMPLE XIV: 5 Demonstration of Antibody-Antibody Interaction in a Protein MicroarrayThe following example illustrates the utility of an embodiment of the present invention for demonstrating the fabrication of an array of biotinylated antibodies on acoated surface of the present invention, and their subsequent detection using antibody-antibody recognition. 10 Surface Fabrication. Glass microscope slides with amine reactive surface chemistries were prepared as described in Example X. Streptavidin was covalentlyattached to the surface using the following procedure. First, an adhesive hybridizationchamber (Schleicher and Schuell) was placed on the glass substrate. Streptavidin(Prozyme, Inc.) was diluted to 100 pg/ml in phosphate buffer (50 mM sodium phosphate, 15 pH 7.0), and approximately 700 μϊ was added to the hybridization chamber, where thereaction was allowed to proceed for 1 hour. The chamber was then removed, and slideswere rinsed and then submerged in a deactivation solution consisting of 50 mMethanolamine in 50 mM phosphate buffer, pH 7. The deactivation was allowed toproceed for 1 hour, at which point the slides were removed from the solution, rinsed with 20 water and dried.

Microarray Printing: Lyophilized biotinylated goat antibody (goat anti-mouseIgG, biotin-labeled, Pierce) was reconstituted to Tmg/ml in phosphate buffered salinewith 15 mg/ml bovine serum albumin (reconstitution buffer as supplied by Pierce). Thisstock was then diluted to 100 pg/ml antibody in phosphate buffered saline with varying 25 amounts of Tween20® surfactant. A 384-well source plate was prepared with 20 μϊdroplets of the antibody solution, with wells dedicated to 0, 0.0001, 0.0005, 0.001, and0.005, 0.01, 0.05 vol% Tween20®. The coating chemistry was first triple-rinsed withultrapure (18 ΜΩ-cm) H?O and then blown dry with N2 gas prior to arraying. An array ofspots was printed on the chemistries using a SpotBot microarrayer (Telechem) fitted with 30 SMP3B pins (Telechem). Spotted slides were incubated at 75% relative humidity for 1.5hours.

Specific Binding Assay: The specific binding assays were performed at roomtemperature as follows: chemistries were incubated with 700 μΐ of 10/xg/ml of Cy3 42 labeled rabbit anti-goat IgG (Sigma) in PBS (0.1 M sodium phosphate, 0.15 M sodiumchloride, pH 7.2), using an adhesive hybridization chamber (described above). Slideswere incubated for 30 minutes. The hybridization chamber was then removed and slideswere triple-rinsed with PBST (0.1 M sodium phosphate, 0.15 M sodium chloride, pH 7.2, 5 0.05% Tween20®) then ultrapure (18 ΜΩ-cm) Η3Ο and dried. Slide chemistries were placed in a GenePix 4000B scanner (Axon) and settings were optimized for the bestsignal to noise performance. Final scanner settings were 100% laser power and 460 voltsetting on the photo multiplier tube.

Binding Residts: A representative array image is presented in Fig. 15. All spots 10 in the image are printed with the same antibody concentration, 100 pg/ml. Each row hasa different Tween20® concentration in the buffer, with Tween20® concentrationincreasing from 0 in the top row to 0.05% in the bottom row. Five duplicate spots areprinted in each row. The first observation is that the arrayed antibodies are recognizedby the fluorescently labeled detection antibody, demonstrating the surface immobilization 15 of the printed antibody. The addition of Tween20® to the print buffer increases both the size and intensity of the printed spots. Spots in the bottom row have specific bindingsignal of approximately 9000 relative fluorescence units (RFUs), while the localbackground is roughly 1000 RFUs. EXAMPLE XV: 20 Demonstration of Synergistic Effect of Coating Components for

Improving Non-Specific Binding Properties of Coated SubstratesThe following example was designed to show that the combination of components embodied in the present invention provide performance superior to coatings comprised ofsubsets of these components. 25 The components in the “standard formula” coating solution can be abbreviated as follows:

Biotin-PEG Biotin-PEG-CCVN-hydroxysuccinimidyl ester (Shearwater Corp.)Aminosilane (3-trimethoxysilylpropyl)-diethylenetriamine (Gelest)

Azidosilane 6-azidosulfonylhexyl-triethoxysilane (Gelest) 30 PST polyoxyethylene sorbitan tetraoleate (Aldrich)

For this experiment, coating solutions were prepared in the standard formula (as described in Example I), as well as in various subtractions and combinations of these 43 components. In all cases, the volume and ratio of carrier solvents (1:4 DMAC:DMSO)was held constant.

Glass microscope slides were coated as described in Example V.

The surfaces were then assayed for non-specific binding using the procedure5 described in Example II.

Binding Results: Results are presented in Fig. 16. Results show that the standardformulation provides the best NSB performance relative to all other componentcombinations, with the exception of the “no azidosilane” formulation, which showsequivalent NSB in this assay. Subsequent experiments have shown that the “no 10 azidosilane” formulation has inferior shelf life relative to the standard formulation.

Specifically, once the surface is exposed to water rinse, the low NSB properties of the “noazidosilane” degrade much more rapidly than the standard formulation (days versusweeks or months). This is consistent with the concept that the azidosilane provides somecross-linking/stabilizing role in the surface coating. 15 The data from this example shows that embodiments of the present invention operate at optimal performance when all the components of the surface coating arepresent. EXAMPLE XVI:

Demonstration of Additional Matrix Forming Components in the Coating 20 Formulation

The following example illustrates the utility of an embodiment of the present invention utilizing alternative matrix-forming molecules, as compared to polyoxyethylenesorbitan tetraoleate (PST). The experiments described here demonstrate otherembodiments of the invention, in which alternative matrix-forming molecules provide 25 good non-specific binding and specific binding performance.

Coating solutions were prepared as described in Example Π. In addition, coating solutions were also prepared in which the PST was replaced with commercial surfactants,namely Triton X-100 and Tween20. As a control, the matrix-forming component wasreplaced with glycerol in the formulation. Note that glycerol is not expected to show 30 equivalent matrix-forming ability.

Coating solutions were cast on silicon wafers and cured as described in Example II. The non-specific binding assay was performed as described in Example II. Thespecific binding assay was performed as described in Example IX. 44 w

Binding Results: Results are presented in Fig. 17. Fig. 17A shows that all threematrix-forming components outperform BSA-blocking in the NSB experiment. Of thethree tested, the PST gives the best performance, followed by Tween20 and Triton X-100.As expected, coatings made with the glycerol substitute had very high NSB. 5 Fig. 17B shows that specific binding performance is similar for all three matrix- forming components. Although the glycerol also gives high signal, this is most likely theresult of non-specific, rather than specific binding, as suggested by the NSB experiment. EXAMPLE XVII:

Inhibition of Cell Proliferation on Coated Surfaces10 The following example illustrates the inhibition of mammalian cell proliferation from culture on one embodiment of the present invention. Coatings were prepared ontissue culture polystyrene substrates in the manner described in Example HI.

Cell Culture: Routine cell culture methods using sterile technique and commercialcell culture media and cell lines were employed. Two cell lines were employed (L929 15 fibroblasts, ATCC, and human umbilical venous endothelial cells, HUVECs,BioWhittaker). Cell culture media for L929 culture, changed every three days,comprised commercial 10% fetal bovine serum in DMEM saline with antibioticsupplements. The control surface was a sterile uncoated tissue culture polystyrene petridish (BD-Falcon) with cells subject to identical conditions. Cultured stock cells 20 trypsinized from commercial culture ware after reaching confluent phase were plated at adensity of 105 per dish and incubated in a commercial cell incubator under 5% CO2 at37°C. -

Results: Fig. 18 provides microscope images of the L929 fibroblast cell culturesurfaces after three days of growth. The coated surface Fig. 18A shows few cells relative 25 to the TCPS control Fig. 18B, which shows adherent cell monolayer coverage typical ofculture for these cells. The cells that are present on the coated surface appear rounded, inloosely bound clusters, clearly avoiding surface contact and unable to generate confluentcell monolayers or consistent cell attachment. Their rounded shape indicates a stressedcondition for these attachment-dependent cells. The control surface shows spread, 30 adherent cells typically observed for these cultures of viable cells. It is believed that theinhibition of extracellular matrix protein adhesion to the coated surface inhibits fibroblastproliferation. Bacterial adhesion and proliferation was also investigated in preliminaryexperiments. Pseudomonas aeruginosa strain PA01 (ATCC, Manassas, VA) cultured in 45 tryptic soy broth (TSB) per routine microbiological methods to a culture density of ~109CFU/ml and inoculated onto coated polystyrene petri dishes in various dilutions from thisstock using TSB. Bacteriological grade polystyrene (BD) was used a control surface.After 24-hour incubation in a culture incubator (37°C) and rinsing with TSB, few 5 adherent microbes were observed on the coated surface using phase contrast microscopy(40x magnification). By contrast, the control surface was colonized with adherent viableorganisms to high density, not removable by media rinsing (data not shown).

It will be clear that the invention is well adapted to attain the ends and advantagesmentioned as well as those inherent therein. While a presently preferred embodiment has 10 been described for purposes of this disclosure, various changes and modifications may bemade which are well within the scope of the invention. Numerous other changes may bemade which will readily suggest themselves to those skilled in the art and which areencompassed in the spirit of the invention disclosed herein and as defined in the appendedclaims. 15 All publications cited herein are hereby incorporated by reference. 46