Modification of solid supports with biomolecules (nucleic acids, peptides) finds application in sensing, chromatography, medical diagnostics, and related areas where specific recognition between immobilized and free species provides diagnostic information or serves as part of a purification or separations process. The chemistry employed for immobilization of biomolecules to the solid support is of crucial importance as it impacts the activity and permanence of the surface-tethered layer. This report considers preparation of maleimide-activated siliceous surfaces, which are often employed for surface conjugation of thiolated biomolecules. Emphasis is placed on validation of each chemical step involved. Use of high surface area fumed silica as the solid support enabled application of standard analytical techniques of infrared spectroscopy, elemental analysis, and titration assays to the modified surfaces. Silica derivatized with (aminopropyl)triethoxysilane (APTES) in a first step was then activated with heterobifunctional cross-linkers to introduce maleimide groups. The cross-linkers bear an amine-reactive site, either isocyanate or N-hydroxysuccinimide ester (NHS-ester), in addition to a thiol-reactive maleimide. In applications, the intention is for the amine-reactive site to react with the surface (e.g., APTES) amines while retaining maleimide activity for subsequent reaction with thiols (e.g., on a biomolecule). Isocyanate cross-linkers are found to yield surfaces highly active in maleimide groups and are used to demonstrate subsequent immobilization and hybridization of DNA oligonucleotides. In contrast, under comparable conditions, NHS-ester cross-linkers were found to be less effective due to deactivation of their maleimide groups by side reaction with surface amines. The coverage of each species, APTES and cross-linker, and thus the stoichiometry of their reaction were also determined.
|Original language||English (US)|
|Number of pages||8|
|State||Published - Aug 19 2003|
ASJC Scopus subject areas
- Materials Science(all)
- Condensed Matter Physics
- Surfaces and Interfaces