Tunable graphene quantum dot surface chemistry enables fast and selective detection of viral RNA

Fast and reliable detection of viral infections is essential for effective disease control, early intervention, and the prevention of widespread outbreaks. In this context, we propose a homogeneous fluorescence-based assay using graphene quantum dots (GQDs) as a biosensing platform for the rapid and selective detection of viral nucleic acid sequences, demonstrated here with SARS-CoV-2 as a model target. A FAM-labelled single-stranded oligonucleotide, specific to the SARS-CoV-2 RdRP sequence, was immobilized onto the surface of GQDs via non-covalent interactions. We investigated the impact of surface oxygen functionalities on assay performance using three GQD variants: oxidized GQDs (GQD-Ox), carboxyl-functionalized GQDs (GQD-COOH), and hydroxyl-functionalized GQDs (GQD-OH). Upon hybridization with the complementary target, fluorescence quenching was observed, while negligible changes were seen with non-complementary and mutated sequences-confirming the assay's high selectivity. A strong linear correlation between fluorescence signal and target concentration was established across a wide dynamic range for all GQDs, with GQD-Ox exhibiting superior performance in terms of sensitivity, selectivity, linearity, and reproducibility. Importantly, the assay was finally optimized to reduce the target incubation time from 30 min to just 5 min, enabling the detection of SARS-CoV-2 sequences at concentrations as low as 139 pg/μL. These findings demonstrate the potential of GQD-Ox as a robust and efficient platform for the development of rapid, sensitive, and selective genosensors, with applicability not only to SARS-CoV-2 but also to other viral pathogens of clinical concern.