Probing of EDC/NHSS-Mediated Covalent Coupling Reaction by the Immobilization of Electrochemically Active Biomolecules

1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide / N-hydroxysulfosuccinimide (EDC/NHSS)-mediated two-step amine coupling reaction is one of most attractive approaches for the elaboration of affinity biosensors. In this work, we investigated the EDC/NHSS-mediated reaction on 3-mercaptopropionic acid (MPA)-covered electrode by the immobilization of electrochemically active biomolecules (dopamine or DA, amyloid-β–Cu2+ or Aβ–Cu2+ and Laccase). The amino groups of DA molecules and lysine residues of Aβ/Laccase make these analytes easy for the reaction with EDC/NHSS-activated MPA self-assembled monolayer (SAM). Effects of EDC/NHSS concentration ratio, pH value, activation/coupling time and analyte concentration on the coupling efficiency were investigated by cyclic voltammetry. The optimal conditions for the EDC/NHSS-mediated amine coupling reaction were reported. We believe that this work will be valuable for the immobilization of biomolecules and the fabrication of affinity biosensors.