Free-standing three-dimensional graphene scaffolds for protease functional assay

Abstract

Three-dimensional graphene scaffolds (3d-GS) of high porosity possessing good fluorescence quenching properties are potential candidates for the development of optical biosensors. Herein, we demonstrate the feasibility of utilising intact and free-standing 3d-GS for sensitive detection of proteases, a class of disease diagnosis biomarkers of significant interest. Recombinant OmpT was employed as a model protease for validating the proposed methodology. A short (15-residue) peptide sequence encoding a specific recognition site for OmpT was end-labelled with a fluorescent dye (5-FAM) whose fluorescence is quenched when the peptide is anchored to 3d-GS. However, in the presence of OmpT, the peptide is cleaved and released from 3d-GS, resulting in a significant recovery in fluorescence. The functional assay described herein involves a single step fabrication process of anchoring the peptide to 3d-GS. The integrity of the 3d-GS is hypothesised to overcome the concern of dynamic re-quenching associated with the typical homogeneous assays based on graphene, yielding a limit of detection (LOD) of ∼ 140 nM, which is over an order higher than homogeneous assays performed using the same composition of graphene in powdered form. To the best of our knowledge, this is the first report on utilising free-standing 3d-GS for facile assaying of proteases. © 2023 Elsevier B.V.