Carbodiimide/NHS Derivatization of COOH-Terminated SAMs: Activation or Byproduct Formation?

COOH-terminated self-assembled monolayers (SAMs) are widely used in biosensor technology to bind different amine-containing biomolecules. A covalent amide bond, however, can be achieved only if the carboxylic acids are activated. This activation process usually consists of forming an <i>N</i>-hydroxysuccinimidyl ester (NHS-ester) by consecutively reacting carboxylic acids with a carbodiimide and NHS. Though many papers report using this method,− the experimental conditions vary greatly between them and chemical characterization at this stage is often omitted. Evidence of an efficient activation is therefore rarely shown. Furthermore, recent publications− have highlighted the complexity of this process, with the possible formation of different byproducts. In this paper, we have conducted a study on NHS activation under different conditions with chemical characterization by polarization-modulation infrared reflection–absorption spectroscopy (PM-IRRAS) and time-of-flight secondary ion mass spectroscopy (ToF-SIMS). Our results indicate that the nature of the solvent and carbodiimide and the reactant concentrations play crucial roles in activation kinetics and efficiency