Specific Chemical Modification of Nanohole Edges in Membrane Graphene for Protein Binding

The vital role of biosensors in our lives is steadily increasing due to their wide range of applications. As part of our striving efforts to develop affordable and highly sensitive biosensing technologies, we present here the successful chemical modification of─and biological molecule attachment to─holes’ edges formed in a sheet of graphene, named nanomembrane graphene (NMG). This work complements our previous work, which showed that NMG could be used as a mid-IR biosensor, in which it becomes essential to overcome the challenge of the specific chemical modification of the holes’ edges. In this work, we formed the NMG on reduced graphene oxide (rGO) layer using Au nanoparticles (Au NPs) and nano-islands (Au NIs). The formation methods were optimized by applying a matrix of variable concentration, size, and deposition time, as well as by chemical modification of substrate. The optimum scenarios were defined as having an extremely thin rGO layer, Au NPs, or NIs with size and center-to-center distance of 20–35 and 40–60 nm, respectively, and to have weak interaction between the metal and the substrate to allow etching leading to the formation of holes. The chemical groups at the edges were investigated to define the best method to attach biological molecules to them. Finally, we demonstrated the successful measurement of the binding between SARS-CoV-2 spike protein and its antibody (ACE2); real-time binding measurements revealed an affinity constant of 0.93 × 109 M–1. We consider these results important as they demonstrate a new route to a low-cost and high-sensitivity biosensor.