This paper reports a convenient method for immobilizing biologically active ligands to self-assembled monolayers of alkanethiolates on gold (SAMs). This methodology is based on monolayers that present maleimide and penta(ethylene glycol) groups. The maleimide groups react efficiently with thiol-terminated ligands, whereas the penta(ethylene glycol) groups prevent the nonspecific adsorption of protein to the substrate. The rate and selectivity of the immobilization of a ferrocene-thiol conjugate were characterized using cyclic voltammetry. This paper presents three examples of biochips prepared using this methodology. In the first example, four carbohydrate-thiol conjugates were immobilized to monolayers and the lectinbinding properties of the substrates were examined using fluorescence and surface plasmon resonance spectroscopy. The second biochip was used to study the enzymatic phosphorylation of the immobilized peptide IYGEFKKKC by the tyrosine kinase c-src. Monolayers presenting this peptide were then used to study the inhibition of the enzyme in an array format. The final class of substrates, which presents the tripeptide Arg-Gly-Asp, was used for studies of integrin-mediated cell adhesion. The immobilization methodology described here, which combines the structural order and inert properties of SAMs with the efficient reaction between soluble thiol and surface-bound maleimide groups, will be useful for preparing substrates for a wide range of applications in basic science and biotechnology. † Part of the Langmuir special issue entitled The Biomolecular Interface.

Alkanephosphonic acids assemble from solution on the native oxide surface of titanium to form alkane chain ordered films which, however, are easily removed by solvent rinse. In contrast, assembling the alkanephosphonic acid from solution on the native oxide surface of titanium followed by gentle heating gives an alkane chain ordered film of the acid which is strongly surface-bound; this film resists removal by solvent washing or simple mechanical peel testing. Surface imaging by atomic force microscopy shows comprehensive surface coverage by aggregated islands, with elevations of each consistent with monolayer formation. ω-Carboxyalkanephosphonic acids are also easily bound to the native oxide surface of Ti through self-assembly/heating. Infrared analysis is consistent with surface attachment through the phosphonate headgroup.

Phosphonate-steel interactions have been industrially significant for decades, but details of the phosphonate-steel interface have not yet been characterized. Self-assembled monolayers of phosphonic acids were formed on stainless steel 316L by room-temperature solution deposition. The acids are covalently bound to the surface as phosphonates in a bidentate manner, as determined by diffuse reflectance Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. Complete coverage of the surface is confirmed by contact angle measurement and atomic force microscopic imaging. This method of monolayer formation contrasts the requirement for heating and long reaction times found to be necessary to form phosphonate monolayers on other metal oxide substrates, such as titanium and silicon.

Experimental SectionGeneral. Aminopropyl(triethoxy)silane (1) (Gelest), 3-maleimidopropionic acid N-hydroxysuccinimide ester (3) (Aldrich, 99%), and RGDC (American Peptide) were used as received. Acetonitrile was distilled from CaH2.Titanium(III) Dihydrogen Orthophosphate on Ti-6Al-4V (TiP). Disks of Ti-6Al-4V ELI ( 3 /8 in. diameter) were sanded first with 240 grit and then 600 grit silicon carbide paper, rinsed, and sonicated successively with methylene chloride, methyl ethyl ketone, and methanol, dried, and stored in an oven at 200 °C. The disks were placed in a solution of aqueous phosphoric acid (1.4 M) as previously described for pure Ti. 16 TiP was identified as the sole surface species present, by X-ray diffraction analysis.Attachment of RGD to Ti-6Al-4V ELI via the TiP Interface. Disks of Ti-6Al-4V ELI coated with TiP were placed in a Teflon well, treated with a solution of 1 (10 mM in THF), and then solvent rinsed with sonication to give surface-bound 2 (IR: νCH 2 ) 2925, 2860 cm -1 ; residual νOH ) 3200 cm -1 ; νNH 2 group ) 1575 cm -1 9 ). The disks were then placed in solutions of 3 (5 mM in acetonitrile) for 18 h at room temperature. The disks were removed from solution, solvent evaporated, and analyzed by IR. They were then rinsed in acetonitrile with sonication and dried in vacuo (0.1 Torr) to give surface-bound 4 (IR: νCO(asymm) ) 1709 cm -1 ; absorbance intensity ≈0.1% vs ≈0.003% for the adduct prepared by direct silanization of the native oxide 9 ). A solution of RGDC (5 mM) was prepared in 5 mL of purified water (Millipore), with the pH adjusted to 6.5 using 0.1 M NaOH. Disks of 4 were left in the stirred solution of the peptide at room temperature for 24 h to give surface bound RGDC (5). They were then removed, dried, and analyzed by IR. The disks were then rinsed with water, dried, subjected to tape peel testing, and reanalyzed by IR (for 5, νCO ) 1700, 1650, 1560 (shoulder) cm -1 9 ).Attachment of RGD to Ti-6Al-4V ELI via an 11-Hydroxyundecylphosphonate Interface. Disks of Ti-6Al-4V were placed in a Teflon well and covered with a solution of