Received July 27, 2000

In Final Form: January 16, 2001

Abstract:

A novel surface modification procedure for the creation of sulfhydryl-terminated alkanethiol monolayers that can be used for the attachment of biomolecules onto gold surfaces is described. A self-assembled monolayer of the amine-terminated alkanethiol 11-mercaptoundecylamine (MUAM) is reacted with the heterobifunctional cross-linker N-succinimidyl S-acetylthiopropionate (SATP) in order to create a protected sulfhydryl-terminated monolayer. This monolayer can then be deprotected in an alkaline solution to create an active sulfhydryl surface. Compounds that have been modified to contain a maleimide moiety can be easily attached onto the sulfhydryl-derivatized gold surface. In a second attachment strategy, the sulfhydryl-terminated monolayer is reacted with 2,2'-dipyridyl disulfide to form disulfide bonds on the surface. These disulfide bonds are then used in a thiol-disulfide exchange reaction with free sulfhydryls in order to attach biomolecules, such as thiol-modified DNA or cysteine-containing polypeptides, onto the surface. In contrast to the maleimide-attached monolayers, the disulfide-immobilized species can be cleaved in the presence of dithiothreitol (DTT) in order to regenerate the free sulfhydryl surface. Polarization modulation FTIR reflection-absorption spectroscopy (PM-FTIRRAS) has been used to characterize these surface reactions, and fluorescence "wash off" measurements provided an estimate of 1.5 × 10¹² molecules/cm² for the surface coverage of DNA immobilized using a thiol-disulfide exchange reaction. Surface plasmon resonance (SPR) imaging measurements were employed to monitor in situ hybridization onto DNA arrays fabricated using this surface immobilization reaction.

Download the full text: PDF | HTML