DNA Hybridization\nKinetics Observed at the Single-Molecule\nLevel Using Graphene Near-Field Effects

We present the development of an advanced sensing platform\nusing\na monolayer of graphene functionalized with fluorophore-labeled DNA\nhairpins to detect the kinetics of single hairpins during the hybridization\nreaction. The near-field photonic effects of graphene induce a distance-dependent\nquenching effect on the attached fluorescent labels, resulting in\ndistinct optical signals in response to axial displacements resulting\nfrom DNA hybridization. Employing a wide-field Total Internal Reflection\nFluorescence (TIRF) optical setup coupled with a sensitive Electron-Multiplying\nCharge-Coupled Device (EM-CCD) camera, we successfully detected fluorescent\nsignals of individual or a low number of individual DNA hairpins within\na low-concentration environment DNA target (tDNA). These signals were\nused to determine the optical setup’s Point Spread Function\n(PSF) in a novel approach to super-resolution reconstruction. Combining\nthese techniques, the subpixel localization of single hairpin molecules\nand their respective intensity profiles were extracted, enabling a\nkinetic assessment of individual DNA hairpins, with estimated unfolding\ntimes of approximately 7 s. Observations of kinetic phenomena unveiled\nintermediate partially hybridized states, extending the time required\nto unfold the hairpin probes by more than a factor of 2. Furthermore,\na developed semiempirical model allowed the conversion of fluorescent\nsignals into fluorophore–graphene distances. At the nanometer\nscale, we observed a step-like unfolding process characterized by\nintermittent metastates of unfolding and static periods, which can\nbe attributed to nucleation events in some cases. Our graphene-based\nsensing platform and optical methodologies can be adopted for further\nresearch into the kinetics of different biomolecules under diverse\nenvironmental conditions.