A three-step reaction procedure was applied to introduce RGD-containing peptides on the titanium surface. Water-vapor-plasma-pretreated titanium surfaces were first silanized with (3-aminopropyl)triethoxysilane, resulting in a multilayer film of poly(3-aminopropyl)siloxane. In a second reaction step, the free primary amino groups were linked to one of the three hetero-cross-linkers: N-succinimidyl-6maleimidylhexanoate, N-succinimidyl-3-maleimidylpropionate, and N-succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate. Onto the resulting terminal-maleimide surface, two model, cell-adhesive peptides, H-Gly-Arg-Gly-Asp-Ser-Pro-Cys-OH and H-Arg-Gly-Asp-Cys-OH were immobilized through covalent addition of the cysteine thiol (-SH) group. X-ray photoelectron spectroscopy, infrared reflection absorption spectroscopy, and radiolabeling techniques were applied to characterize the surfaces. From independent quantitative analysis, an approximate coverage of 0.2∼0.4 peptides/nm 2 was calculated.

Infrared spectroscopy was applied to investigate the well-known EDC/NHS (N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide/N-hydroxysuccinimide) activation details of poly(acrylic acid) (PAA) and poly(methacrylic acid) (PMAA) brushes grafted on porous silicon. Succinimidyl ester (NHS-ester) is generally believed to be the dominant intermediate product, conveniently used to immobilize biomolecules containing free primary amino groups via amide linkage. To our surprise, the infrared spectral details revealed that the EDC/NHS activation of PMAA generated anhydride (estimated at around 76% yield and 70% composition), but not NHS-ester (around 5% yield and 11% composition) under the well-documented reaction conditions, as the predominant intermediate product. In contrast, EDC/NHS activation of PAA still follows the general rule, i.e., the expected NHS-ester is the dominant intermediate product (around 45% yield and 57% composition), anhydride the side product (40% yield and 28% composition), under the optimum reaction conditions. The following amidation on PAA-based NHS-esters with a model amine-containing compound, L-leucine methyl ester, generated approximately 70% amides and 30% carboxylates. In contrast, amidation of PAA- or PMAA-based anhydrides with L-leucine methyl ester only produced less than 30% amides but more than 70% carboxylates. The above reaction yields and percentage compositions were estimated by fitting the carbonyl stretching region with 5 possible species, NHS-ester, anhydride, N-acylurea, unreacted acid, unhydrolyzed tert-butyl ester, and using the Beer-Lambert law. The different surface chemistry mechanisms will bring significant effects on the performance of surface chemistry-derived devices such as biochips, biosensors, and biomaterials.

Surface modification of acid-pretreated titanium with 3-aminopropyltriethoxylsilane (APTES) in dry toluene resulted in covalently bonded siloxane films with surface coverage that was relatively controllable by regulating the reaction conditions. A hetero-bifunctional cross-linker, N-succinimidyl-3-maleimidopropionate (SMP), reacted with the terminal amino groups, forming the exposed maleimide groups. Finally, a model cell-binding peptide, Arg-Gly-Asp-Cys (RGDC), was immobilized on the surface through covalent addition of the cysteine thiol groups to the maleimide groups. X-ray photoelectron spectroscopy, radiolabelling techniques, and ellipsometry were used to quantify and characterize the modified surfaces.