"Click" immobilization on alkylated silicon substrates: model for the study of surface bound antimicrobial peptides

We describe an effective approach for the covalent immobilization of antimicrobial peptides (AMPs) to bioinert substrates via Cu(I) -catalyzed azide-alkyne cycloaddition (CuAAC). The bioinert substrates were prepared by surface hydrosilylation of oligo(ethylene glycol) (OEG) terminated alkenes on hydrogen-terminated silicon surfaces. To render the OEG monolayers "clickable", mixed monolayers were prepared using OEG-alkenes with and without a terminal alkyne protected by a trimethylgermanyl (TMG) group. The mixed monolayers were characterized by X-ray photoelectron spectroscopy (XPS), elliposometry and contact angle measurement. The TMG protecting group can be readily removed to yield a free terminal alkyne by catalytic amounts of Cu(I) in an aqueous media. This step can then be combined with the subsequent CuAAC reaction. Thus, the immobilization of an azide modified AMP (N3-IG-25) was achieved in a one-pot deprotection/coupling reaction. Varying the ratio of the two alkenes in the deposition mixture allowed for control over the density of the alkynyl groups in the mixed monolayer, and subsequently the coverage of the AMPs on the monolayer. These samples allowed for study of the dependence of antimicrobial activities on the AMP density. The results show that a relative low coverage of AMPs (～1.6×10(13) molecule per cm(2)) is sufficient to significantly suppress the viability of Pseudomonas aeruginosa, while the surface presenting the highest density of AMPs (～2.8×10(13) molecule per cm(2)) is still cyto-compatible. The remarkable antibacterial activity is attributed to the long and flexible linker and the site-specific "click" immobilization, which may facilitate the covalently attached peptides to interact with and disrupt the bacterial membranes.     

Biofunctionalization of a "clickable" organic layer photochemically grafted on titanium substrates

We have developed a general method combining photochemical grafting and copper-catalyzed click chemistry for biofunctionalization of titanium substrates. The UV-activated grafting of an α,ω-alkenyne onto TiO(2)/Ti substrates provided a "clickable" thin film platform. The selective attachment of the vinyl end of the molecule to the surface was achieved by masking the alkynyl end with a trimethylgermanyl (TMG) protecting group. Subsequently, various oligo(ethylene glycol) (OEG) derivatives terminated with an azido group were attached to the TMG-alkynyl modified titanium surface via a one-pot deprotection/click reaction. The films were characterized by X-ray photoelectron spectroscopy (XPS), contact angle goniometry, ellipsometry, and atomic force microscopy (AFM). We showed that the titanium surface presenting click-immobilized OEG substantially suppressed the nonspecific attachment of protein and cells as compared to the unmodified titanium substrate. Furthermore, glycine-arginine-glycine-aspartate (GRGD), a cell adhesion peptide, was coimmobilized with OEG on the platform. We demonstrated that the resultant GRGD-presenting thin film on Ti substrates can promote the specific adhesion and spreading of AsPC-1 cells.     

Covalently modified silicon and diamond surfaces: resistance to nonspecific protein adsorption and optimization for biosensing

We report the direct covalent functionalization of silicon and diamond surfaces with short ethylene glycol (EG) oligomers via photochemical reaction of the hydrogen-terminated surfaces with terminal vinyl groups of the oligomers, and the use of these monolayers to control protein binding at surfaces. Photochemical modification of Si(111) and polycrystalline diamond surfaces produces EG monolayers linked via Si-C bond formation (silicon) or C-C bond formation (diamond). X-ray photoelectron spectroscopy was used to characterize the monolayer composition. Measurements using fluorescently labeled proteins show that the EG-functionalized surfaces effectively resist nonspecific adsorption of proteins. Additionally, we demonstrate the use of mixed monolayers on silicon and diamond and apply these surfaces to control specific versus nonspecific binding to optimize a model protein sensing assay.     

Functional monolayers for improved resistance to protein adsorption: oligo(ethylene glycol)-modified silicon and diamond surfaces

The interaction of proteins with semiconductors such as silicon and diamond is of great interest for applications such as electronic biosensing. We have investigated the use of covalently bound oligo(ethylene glycol), EG, monolayers on diamond and silicon to minimize nonspecific protein adsorption. Protein adsorption was monitored by fluorescence scanning as a function of the length of the ethylene glycol chain (EG3 through EG6) and the terminal functional group (methyl- versus hydroxyl-terminated EG3 monolayer). More quantitative measurements were made by eluting adsorbed avidin from the surface and measuring the intensity of fluorescence in the solution. The attachment chemistry of the tri(ethylene glycol) molecules and monolayer orientation was studied by X-ray photoelectron spectroscopy. Improvement in the selectivity of surfaces modified with EG functionality was demonstrated in two model biosensing assays. We find that high-quality EG monolayers are formed on silicon and diamond and that these EG3 monolayers are as effective as EG3 self-assembled monolayers on gold at resisting nonspecific avidin adsorption. These results show promise for use of silicon and diamond materials in many potential applications such as biosensing and medical implants.     

Factors that determine the protein resistance of oligoether self-assembled monolayers --internal hydrophilicity,terminal hydrophilicity,and lateral packing density

Protein resistance of oligoether self-assembled monolayers (SAMs) on gold and silver surfaces has been investigated systematically to elucidate structural factors that determine whether a SAM will be able to resist protein adsorption. Oligo(ethylene glycol) (OEG)-, oligo(propylene glycol)-, and oligo(trimethylene glycol)-terminated alkanethiols with different chain lengths and alkyl termination were synthesized as monolayer constituents. The packing density and chemical composition of the SAMs were examined by XPS spectroscopy; the terminal hydrophilicity was characterized by contact angle measurements. IRRAS spectroscopy gave information about the chain conformation of specific monolayers; the amount of adsorbed protein as compared to alkanethiol monolayers was determined by ellipsometry. We found several factors that in combination or by themselves suppress the protein resistance of oligoether monolayers. Monolayers with a hydrophobic interior, such as those containing oligo(propylene glycol), show no protein resistance. The lateral compression of oligo(ethylene glycol) monolayers on silver generates more highly ordered monolayers and may cause decreased protein resistance, but does not necessarily lead to an all-trans chain conformation of the OEG moieties. Water contact angles higher than 70 degrees on gold or 65 degrees on silver reduce full protein resistance. We conclude that both internal and terminal hydrophilicity favor the protein resistance of an oligoether monolayer. It is suggested that the penetration of water molecules in the interior of the SAM is a necessary prerequisite for protein resistance. We discuss and summarize the various factors which are critical for the functionality of "inert" organic films.     

Nanoscale water condensation on click-functionalized self-assembled monolayers

We have examined the nanoscale adsorption of molecular water under ambient conditions onto a series of well-characterized functionalized surfaces produced by Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC or "click") reactions on alkyne-terminated self-assembled monolayers on silicon. Water contact angle (CA) measurements reveal a range of macroscopic hydrophilicity that does not correlate with the tendency of these surfaces to adsorb water at the molecular level. X-ray reflectometry has been used to follow the kinetics of water adsorption on these "click"-functionalized surfaces, and also shows that dense continuous molecular water layers are formed over 30 h. For example, a highly hydrophilic surface, functionalized by an oligo(ethylene glycol) moiety (with a CA = 34°) showed 2.9 ? of adsorbed water after 30 h, while the almost hydrophobic underlying alkyne-terminated monolayer (CA = 84°) showed 5.6 ? of adsorbed water over the same period. While this study highlights the capacity of X-ray reflectometry to study the structure of adsorbed water on these surfaces, it should also serve as a warning for those intending to characterize self-assembled monolayers and functionalized surfaces to avoid contamination by even trace amounts of water vapor. Moreover, contact angle measurements alone cannot be relied upon to predict the likely degree of moisture uptake on such surfaces.     

Preparation, characterization, resistance to protein adsorption, and specific avidin-biotin binding of poly(amidoamine) dendrimers functionalized with oligo(ethylene glycol) on gold

Protein-resistant films derived from the fifth-generation poly(amidoamine) dendrimers (PAMAM G5) functionalized with oligo(ethylene glycol) (OEG) derivatives consisting of various ethylene glycol units (EG(n), n = 3, 4, and 6) were prepared on the self-assembled monolayers (SAMs) of 11-mercaptoundecanoic acid (MUA) on gold substrates. The resulting films were characterized by ellipsometry, contact angle goniometry, and X-ray photoelectron spectroscopy (XPS). About 35% of the peripheral amines of the dendrimers were reacted with N-hydroxysuccinimide-terminated EG(n) derivatives (NHS-EG(n)). The dendrimer films showed improved stability over octadecanethiolate SAMs on gold in hot solvents, attributed to the formation of multiple amide bonds per PAMAM unit with underlying NHS-activated MUA monolayer. The EG(n)-attached PAMAM surfaces with n = 3 reduced the adsorption of fibrinogen to approximately 20% monolayer, whereas 2-3% for n = 4 or 6. The dendrimer films with various densities of EG(n) molecules on PAMAM surfaces were prepared by immersion of the NHS-terminated MUA-functionalized gold substrates in ethanolic solutions containing PAMAM and NHS-EG(n) of various mole ratios. The density (r) of the EG(n) molecules on the PAMAM surfaces is consistent with the mole ratio (r') of NHS-EG(n)/free amine of PAMAM in solutions. The resistance to protein adsorption of the resulting surfaces is correlated with the surface density and the length of the EG chains. At their respective r, the EG(n)-modified dendrimer films resisted approximately 95% adsorption of fibrinogen on gold surfaces. Finally, the specific binding of avidin to the approximately 5% and approximately 40% biotinylated EG3 dendrimers (surface density of biotin with respect to the total number of terminal amino groups on PAMAM G5) gave rise to about 50% and 100% surface coverage by avidin, respectively.     

Polymer brushes grafted to "passivated" silicon substrates using click chemistry

We present herein a versatile method for grafting polymer brushes to passivated silicon surfaces based on the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition (click chemistry) of omega-azido polymers and alkynyl-functionalized silicon substrates. First, the "passivation" of the silicon substrates toward polymer adsorption was performed by the deposition of an alkyne functionalized self-assembled monolayer (SAM). Then, three tailor-made omega-azido linear brush precursors, i.e., PEG-N3, PMMA-N3, and PS-N3 (Mn approximately 20,000 g/mol), were grafted to alkyne-functionalized SAMs via click chemistry in tetrahydrofuran. The SAM, PEG, PMMA, and PS layers were characterized by ellipsometry, scanning probe microscopy, and water contact angle measurements. Results have shown that the grafting process follows the scaling laws developed for polymer brushes, with a significant dependence over the weight fraction of polymer in the grafting solution and the grafting time. The chemical nature of the brushes has only a weak influence on the click chemistry grafting reaction and morphologies observed, yielding polymer brushes with thickness of ca. 6 nm and grafting densities of ca. 0.2 chains/nm2. The examples developed herein have shown that this highly versatile and tunable approach can be extended to the grafting of a wide range of polymer (pseudo-) brushes to silicon substrates without changing the tethering strategy.     

Different functionalization of the internal and external surfaces in mesoporous materials for biosensing applications using "click" chemistry

We report the use of copper(I)-catalyzed alkyne-azide cycloaddition reaction (CuAAC) to selectively functionalize the internal and external surfaces of mesoporous materials. Porous silicon rugate filters with narrow line width reflectivity peaks were employed to demonstrate this selective surface functionalization approach. Hydrosilylation of a dialkyne species, 1,8-nonadiyne, was performed to stabilize the freshly fabricated porous silicon rugate filters against oxidation and to allow for further chemical derivatization via "click" CuAAC reactions. The external surface was modified through CuAAC reactions performed in the absence of nitrogen-based Cu(I)-stabilizing species (i.e., ligand-free reactions). To subsequently modify the interior pore surface, stabilization of the Cu(I) catalyst was required. Optical reflectivity measurements, water contact angle measurements, Fourier transform infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS) were used to demonstrate the ability of the derivatization approach to selectively modify mesoporous materials with different surface chemistry on the exterior and interior surfaces. Furthermore, porous silicon rugate filters modified externally with the cell-adhesive peptide Gly-Arg-Gly-Asp-Ser (GRGDS) allowed for cell adhesion via formation of focal adhesion points. Results presented here demonstrate a general approach to selectively modify mesoporous silicon samples with potential applications for cell-based biosensing.     

Mixed self-assembled monolayers (SAMs) consisting of methoxy-tri(ethylene glycol)-terminated and alkyl-terminated dimethylchlorosilanes control the non-specific adsorption of proteins at oxidic surfaces

Monolayers from the newly synthesized compound methoxy-tri(ethylene glycol)-undecenyldimethylchlorosilane (CH3O(CH2CH2O)3(CH2)11Si(CH3)2Cl, MeO(EG)3C11DMS) and dodecyldimethylchlorosilane (DDMS), both pure and mixed, were prepared by self-assembly from organic solution in the presence of an organic base. The films obtained were characterized by advancing and receding contact angle measurements and ellipsometry to confirm the formation of self-assembled monolayers (SAMs). The resulting data on the covalently attached dimethylsilanes were compared to known oligo(ethylene glycol) (OEG)-terminated SAM systems based on terminal alkenes, thiolates or trihydrolyzable silanes. The composition of the mixed SAMs was found to depend directly and linearly on the composition of the silanization solution. Enhanced protein repellent properties were found for the SAMs using a variety of proteins, including the Ras Binding Domain (RBD), a protein with high relevance for cancer diagnostics. Roughly a RBD protein monolayer amount was adsorbed to silicon oxide surfaces silanized with DDMS or non-silanized silicon wafers, and in contrast, no RBD was adsorbed to surfaces silanized with MeO(EG)3C11DMS or to mixed monolayers consisting of DDMS and MeO(EG)3C11DMS if the content of OEG-silane overcame a critical content of X(EG) approximately 0.9.     

Novel tertiary amine oxide surfaces that resist nonspecific protein adsorption

Novel surfaces derivatized with tertiary amine oxides have been prepared and tested for their ability to resist nonspecific protein adsorption. The oxidation of tertiary amines supported on triazine units was carried out using mCPBA to give a format allowing conjugation of biologically active ligands alongside them. Adsorption to these surfaces was tested and compared to adsorption to a set of commercial and custom oligo-/poly(ethylene glycol) (OEG/PEG) supports by challenging them with a protein display library presented on bacteriophage lambda. The new class of amine oxide surfaces is found to compare favorably with the performance of the OEG/PEG supports in the prevention of nonspecific binding.     

Monolayer-derivative functionalization of non-oxidized silicon surfaces

This article describes a variety of monolayers anchored directly onto silicon surfaces without an oxide interlayer, their formation mechanisms, their technological applications, and our personal views on the future prospects for this field. The chemical modification of non-oxidized silicon surfaces utilizing monolayers was first reported in 1993. The basic finding that a non-oxidized silicon surface could be neutralized with alkyl chains through direct covalent linkage, i.e., silicon-carbon, has offered chemical scientists ease of handling even in an ambient environment and, thus, research has been predictably focused on forming anti-stiction coating films for nano- and micro-electromechanical systems (NEMS/MEMS). Such surface reforming has also been achieved by using other monolayers, which form interfacial bonds, e.g., silicon-nitrogen and silicon-oxygen. The resultant monolayer surfaces are useful for silicon-based applications including molecular electron transfer films, monolayer templates, molecular insulators, capsulators, and bioderivatives. Such monolayers are applicable not only for surface modification, but also for manipulating individual nanomaterials. By modifying the terminal groups of monolayers with nanomaterials including nanocrystals and biomolecules, the nanomaterials can remarkably be immobilized directly onto non-oxidized silicon surfaces based on the formation mechanisms of the monolayer. Such immobilizations will revolutionize the analysis of the specific features and capabilities of individual nanomaterials. Furthermore, the path will be opened for the development of more advanced monolayer-derived chip technology. To achieve this goal, it is extremely important to thoroughly understand the functionalization processes on silicon, since the resultant internal structures and properties of monolayer-derivative silicon may strongly depend on their course of formation.     

Olefin cross-metathesis of a vinyl-terminated self-assembled monolayer (SAM) on Au(111): electrochemical study using a ferrocenyl redox center

Self-assembled thiol monolayers bound to single-crystal Au(111) surfaces containing a terminal olefin have been prepared and used to monitor electrochemically the cross-metathesis (CM) between the surface and an olefin-terminated ferrocenyl (Fc) derivative from solution over time. Mixed SAM surfaces were prepared by first adsorbing a diluent for 2 days followed by the olefinic alkanethiol for known adsorption time intervals; three diluents of varying length were used. The oxidation peak areas from the voltammetry show the CM reaction yields a maximum amount of product at 100-150 min. Beyond this time, thiol desorption is apparent and the Fc oxidation peaks diminished. A kinetic simulation of the interfacial reactions involving CM and desorption reactions are described and aided in the interpretation of the voltammetric responses. The length of the diluent and the coverage of surface olefins were important factors in limiting undesirable self-CM reactions on the surface, and a model of the relationship between the diluent and surface concentration of olefin is described. This study shows that attention to monolayer formation and reaction conditions are important parameters when maximizing CM yields on surfaces.     

Carboxy-terminated oligo(ethylene glycol)-alkane phosphate: synthesis and self-assembly on titanium oxide surfaces

The surface immobilization of oligo- and poly(ethylene glycol) on solids is a widely used approach to prevent the nonspecific adsorption of proteins, bacteria, and cells. A novel tri(ethylene glycol) derivative, phosphoric acid-mono(22-carboxy-12,15,18,21-tetraoxadocosyl) ester, was synthesized with the aim to produce self-assembled monolayers (SAMs) on metal/metal oxide surfaces. This compound contains two reactive, terminal moieties: the phosphoric acid group as anchor to the surface, and the carboxylic group as linker for further attachment of molecules such as peptides and proteins to be present at the surface. The adsorption on titanium-dioxide-coated substrates was studied quantitatively and the resulting SAMs were characterized by angle-dependent X-ray photoelectron spectroscopy (XPS) and spectroscopic ellipsometry. XPS data showed that the monomolecular layer is attached with the phosphate group to the substrate, but not fully ordered. The dry adlayer thickness was determined to be 13.4 A, which is less than expected for a densely packed monolayer. Surface concentration calculated from ellipsometry data resulted in a grafting density of 2.03 molecules/nm2.     

Covalent biofunctionalization of silicon nitride surfaces

Covalently attached organic monolayers on etched silicon nitride (SixN4; x >/= 3) surfaces were prepared by reaction of SixN4-coated wafers with neat or solutions of 1-alkenes and 1-alkynes in refluxing mesitylene. The surface modification was monitored by measurement of the static water contact angle, XPS, IRRAS, AFM, and ToF-SIMS, and evidence for the formation of Si-C bonds is presented. The etching can be achieved by dilute HF solutions and yields both Si-H and N-H moieties. The resulting etched SixN4 surfaces are functionalized by terminal carboxylic acid groups in either of two ways: (a) via attachment of a 10-undecenoic acid 2,2,2-trifluoroethyl ester (trifluoro ethanol ester) and subsequent thermal acid hydrolysis; (b) through attachment of a photocleavable ester, and subsequent photochemical cleavage, as this would allow photopatterned functionalized SixN4. The carboxylic acids are successfully used for the attachment of oligopeptides (aspartame) and complete proteins using EDC/NHS chemistry. Finally, an amino-terminated organic monolayer can be formed by reaction of HF-treated SixN4 surfaces with a N-(omega-undecylenyl)phthalimide, which yields an amino-terminated surface upon deprotection with hydrazine.     

Engineering silicon oxide surfaces using self-assembled monolayers

Although a molecular monolayer is only a few nanometers thick it can completely change the properties of a surface. Molecular monolayers can be readily prepared using the Langmuir-Blodgett methodology or by chemisorption on metal and oxide surfaces. This Review focuses on the use of chemisorbed self-assembled monolayers (SAMs) as a platform for the functionalization of silicon oxide surfaces. The controlled organization of molecules and molecular assemblies on silicon oxide will have a prominent place in "bottom-up" nanofabrication, which could revolutionize fields such as nanoelectronics and biotechnology in the near future. In recent years, self-assembled monolayers on silicon oxide have reached a high level of sophistication and have been combined with various lithographic patterning methods to develop new nanofabrication protocols and biological arrays. Nanoscale control over surface properties is of paramount importance to advance from 2D patterning to 3D fabrication.     

Structure and reactivity of mixed omega-carboxyalkyl/alkyl monolayers on silicon: ATR-FTIR spectroscopy and contact angle titration

The structure, reactivity, and acid-base properties of mixed monolayers prepared by photochemical reaction of hydrogen-terminated silicon with mixtures of ethyl undecylenate and n-alkenes were studied by ATR-FTIR spectroscopy and contact-angle measurements. The surface composition of the mixed monolayers and its correlation with the hydrolysis reactivity of terminal ethoxycarbonyl (ester) groups were investigated by systematically varying the mole fraction of ethyl undecylenate and the chain length of the unsubstituted alkenes in the binary deposition solution. It has been shown that the mole fraction of ester groups on the surface deviates only slightly from the mole fraction of ethyl undecylenate in the solution. The efficiency of ester hydrolysis under acidic conditions is significantly influenced by the monolayer structure, i.e., the surface density of ester groups and length of the unsubstituted alkyl chains. In addition, we find that mixed omega-alkanoic acid/alkyl monolayers on silicon (prepared via hydrolysis) exhibit well-defined contact angle titration curves from which the surface acid dissociation constants were determined. The results were compared with the acid-base properties reported in the literature for carboxylic acid-terminated alkylsiloxane monolayers on hydroxylated silicon and for omega-mercaptoalkanoic acid/alkanethiolate monolayers on gold. The weak pKa dependence (deltapKa approximately 1) on the surface density of carboxylic acid groups and on the length of unsubstituted alkyl chains is attributed to variations of the microenvironment of the acid moieties. These experimental findings provide fundamental knowledge at the molecular level for the preparation of bioreactive surfaces of controlled reactivity on crystalline semiconductor substrates.     

2D assembly of non-interacting magnetic iron oxide nanoparticles via"click" chemistry

Azide-terminated magnetic iron oxide nanoparticles have been assembled in 2D on alkyne-terminated self-assembled monolayers (SAMs) by the copper(i) catalyzed alkyne-azide cycloaddition (CuAAC) "click" reaction; the kinetics of the reaction is an important parameter to control the interparticle distance and thus the dipolar interactions.     

Poly(ethylene glycol) monolayer formation and stability on gold and silicon nitride substrates

Poly(ethylene glycol) (PEG) self-assembled monolayers (SAMs) are extensively used to modify substrates to prevent nonspecific protein adsorption and to increase hydrophilicity. X-ray photoelectron spectroscopy analysis, complemented by water contact angle measurements, is employed to investigate the formation and stability upon aging and heating of PEG monolayers formed on gold and silicon nitride substrates. In particular, thiolated PEG monolayers on gold, with and without the addition of an undecylic spacer chain, and PEG monolayers formed with oxysilane precursors on silicon nitride have been probed. It is found that PEG-thiol SAMs are degraded after less than two weeks of exposure to air and when heated at temperatures as low as 120 degrees C. On the contrary, PEG-silane SAMs are stable for more than two weeks, and fewer molecules are desorbed even after two months of aging, compared to those desorbed in two weeks from the PEG-thiol SAMs. A strongly bound hydration layer is found on PEG-silane SAMs aged for two months. Heating PEG-silane SAMs to temperatures as high as 160 degrees C improves the quality of the monolayer, desorbing weakly bound contaminants. The differences in stability between PEG-thiol SAMs and PEG-silane SAMs are ascribed to the different types of bonding to the surface and to the fact that the thiol-Au bond can be easily oxidized, thus causing desorption of PEG molecules from the surface.     

Formation of tetra(ethylene oxide) terminated Si-C linked monolayers and their derivatization with glycine: an example of a generic strategy for the immobilization of biomolecules on silicon

Surface modification with oligo(ethylene oxide) functionalized monolayers terminated with reactive headgroups constitutes a powerful strategy to provide specific coupling of biomolecules with simultaneous protection from nonspecific adsorption on surfaces for the preparation of biorecognition interfaces. To date, oligo(ethylene oxide) functionalized monolayer-forming molecules which can be activated for attachment of biomolecules but which can selectively form monolayers onto hydrogen terminated silicon have yet to be developed. Here, self-assembled monolayers (SAMs) containing tetra(ethylene oxide) moieties protected with tert-butyl dimethylsilyl groups were formed by thermal hydrosilylation of alkenes with single-crystal Si(111)-H. The protection group was used to avoid side reactions with the hydride terminated silicon surface. Monolayer formation was carried out using solutions of the alkene in the high-boiling-point solvent 1,3,5-triethylbenzene. The protecting group was removed under very mild acidic conditions to yield a free hydroxyl functionality, a convenient surface moiety for coupling of biological entities via carbamate bond formation. The chemical composition and structure of the monolayers before and after deprotection were characterized by X-ray photoelectron spectroscopy (XPS) and X-ray reflectometry. To demonstrate the utility of this surface for covalent modification, two reagents were compared and contrasted for their ability to activate the surface hydroxyl groups for coupling of free amines, carbonyl diimidazole (CDI), and disuccinimidyl carbonate (DSC). Analysis of XP spectra before and after activation by CDI or DSC, and after subsequent reaction with glycine, provided quantitative information on the extent of activation and overall coupling efficiencies. CDI activated surfaces gave poor coupling yields under various conditions, whereas DSC mediated activation followed by aminolysis at neutral pH was found to be an efficient method for the immobilization of amines on tetra(ethylene oxide) modified surfaces.