DNA Hybridization Kinetics Observed at the Single-Molecule Level Using Graphene Near-Field Effects

We present the development of an advanced sensing platform using a monolayer of graphene functionalized with fluorophore-labeled DNA hairpins to detect the kinetics of single hairpins during the hybridization reaction. The near-field photonic effects of graphene induce a distance-dependent quenching effect on the attached fluorescent labels, resulting in distinct optical signals in response to axial displacements resulting from DNA hybridization. Employing a wide-field Total Internal Reflection Fluorescence (TIRF) optical setup coupled with a sensitive Electron-Multiplying Charge-Coupled Device (EM-CCD) camera, we successfully detected fluorescent signals of individual or a low number of individual DNA hairpins within a low-concentration environment DNA target (tDNA). These signals were used to determine the optical setup's Point Spread Function (PSF) in a novel approach to super-resolution reconstruction. Combining these techniques, the subpixel localization of single hairpin molecules and their respective intensity profiles were extracted, enabling a kinetic assessment of individual DNA hairpins, with estimated unfolding times of approximately 7 s. Observations of kinetic phenomena unveiled intermediate partially hybridized states, extending the time required to unfold the hairpin probes by more than a factor of 2. Furthermore, a developed semiempirical model allowed the conversion of fluorescent signals into fluorophore-graphene distances. At the nanometer scale, we observed a step-like unfolding process characterized by intermittent metastates of unfolding and static periods, which can be attributed to nucleation events in some cases. Our graphene-based sensing platform and optical methodologies can be adopted for further research into the kinetics of different biomolecules under diverse environmental conditions.