Cyclooctyne-Functionalized N-Heterocyclic Carbenes for Click Coupling of Sensing Oligonucleotides to Gold Surfaces

Abstract

Nucleic acid-based sensors (NBEs) are used for biomolecular detection and can enable continuous, real-time molecular monitoring in vivo. NBEs are typically constructed via thiol self-assembled monolayers (SAMs) on gold electrodes, consisting of redox-reporter-modified oligonucleotides diluted within an alkylthiol monolayer. However, the limited stability of thiol SAMs when chronically exposed to biological fluids restricts the long-term in vivo lifespan of NBEs. In contrast, N-heterocyclic carbenes (NHCs) are monolayer-forming ligands that offer improved surface stability versus thiols for biological applications, but to date, the synthetic routes to attach biomolecules to NHC surfaces rely solely on azides tethered to the NHC. As an alternative, employing strain-promoted azide-alkyne cycloadditions (SPAAC) type building blocks on the NHCs, including bicyclo[6.1.0]nonyne (BCN) and dibenzoazacyclooctyne (DBCO), allows for a broader array of azide-modified biomolecules to be employed. In this study, three new masked NHCs for the SPAAC Click reactions were synthesized and characterized. These SPAAC-modified NHCs successfully clicked and transformed their cyclooctynes into triazoles with organic azides at room temperature. The best NHC of the three was deposited onto gold electrode surfaces and successfully achieved coupling of redox-reporter-modified aptamer oligonucleotides, as verified via the electrochemical reduction of their reporter, methylene blue. While these results confirm the success of using cyclooctyne-based NHCs and azide-modified aptamers for copper-free SPAAC Click reactions, more research is needed to improve NHC packing to prevent the generation of hydrogen peroxide on gold during electrochemical interrogation of NBEs. Nevertheless, this study opens novel opportunities for surface modification with an expansive array of aptamers and, potentially, other biomolecules for electrochemical sensing applications.

Keywords: N-heterocyclic carbenes; SPAAC; copper-free Click reactions; electrochemical sensors; nucleic acid−based sensors; surface modification.