Activity analysis of the carbodiimide-mediated amine coupling reaction on self-assembled monolayers by cyclic voltammetry

Abstract

Carbodiimide provides one of the most popular and versatile methods for the covalent attachment of proteins, nucleic acids and small-molecule organic compounds to self-assembled monolayers (SAMs). In this work, we investigated the carbodiimide-mediated amine coupling reaction on SAMs with cyclic voltammetry. The negatively charged 11-mercaptoundecanoic acid (MUA) SAMs on gold electrodes hindered access of the [Fe(CN)6]3−/4− redox probe to the electrode surface. Once the carboxyl groups of MUA were activated by carbodiimide to form O-acylisourea intermediates, the electrostatic interaction between the positively charged intermediates and the negatively charged [Fe(CN)6]3−/4− probes decreased the electron-transfer resistance, resulting in the occurrence of reduction and oxidation reactions of the probe. Hydrolysis and aminolysis of the intermediates induced the loss of the positively charged carbodiimide moieties, causing non-conductive SAMs. Moreover, when the carboxyl groups were converted to amine-reactive N-hydroxysulfosuccinimide (NHSS) ester intermediates, the negatively charged NHSS esters separated from the negatively charged [Fe(CN)6]3−/4− probes and formed a barrier for the electron transfer. The introduction of positively charged amine groups through the amine coupling reaction between an NHSS ester and ethanediamine would facilitate the electron transfer again. With this method, the activity of carbodiimide-mediated activation and acylation reactions and the stability of the resulting O-acylisourea/NHSS ester intermediates in an aqueous system were addressed. The effects of the solution pH and amine concentration on these reactions were also investigated. We believe that the results will be valuable for the immobilization of biomolecules and the fabrication of biosensors.