Hexagonal boron nitride (hBN) is gaining increasing attention in the field of biomolecule characterization due to its compatibility with single-molecule fluorescence imaging and real-time tracking. Embedding fluorescent molecules within hBN layers offers potential for molecular-resolution sensing devices, since these probes are highly sensitive to their surroundings. Yet, the effect of hBN surfaces on the fluorophore properties remains largely unexplored. Here, we monitor the photophysical properties of ATTO647N-ssDNA on hBN surfaces and elucidate the effects of the environment and substrate. We demonstrate that the presence of hBN increases the photobleaching time and changes intermittency dynamics. By combining van der Waals stacking and FDTD simulations, we subsequently engineer hBN optical cavities to modulate the emission from individual molecules, showing that the brightness can be tuned by a factor of 4. Our findings shed light on light–matter interactions in hybrid nanostructures, which can enable single-molecule imaging and biosensing at high spatial and temporal resolution.

Biological nanopores crucially control the import and export of biomolecules across lipid membranes in cells. They have found widespread use in biophysics and biotechnology, where their typically narrow, fixed diameters enable selective transport of ions and small molecules, as well as DNA and peptides for sequencing applications. Yet, due to their small channel sizes, they preclude the passage of large macromolecules, e.g., therapeutics. Here, the unique combined properties of DNA origami nanotechnology, machine-inspired design, and synthetic biology are harnessed, to present a structurally reconfigurable DNA origami MechanoPore (MP) that features a lumen that is tuneable in size through molecular triggers. Controllable switching of MPs between 3 stable states is confirmed by 3D-DNA-PAINT super-resolution imaging and through dye-influx assays, after reconstitution of the large MPs in the membrane of liposomes via an inverted-emulsion cDICE technique. Confocal imaging of transmembrane transport shows size-selective behavior with adjustable thresholds. Importantly, the conformational changes are fully reversible, attesting to the robust mechanical switching that overcomes pressure from the surrounding lipid molecules. These MPs advance nanopore technology, offering functional nanostructures that can be tuned on-demand – thereby impacting fields as diverse as drug delivery, biomolecule sorting, and sensing, as well as bottom-up synthetic biology.

Fluorescence imaging is an invaluable tool to investigate biomolecular dynamics, mechanics, and interactions in aqueous environments. Two-dimensional materials offer large-area, atomically smooth surfaces for wide-field biomolecule imaging. Despite the success of graphene for on-chip biosensing and biomolecule manipulation, its strong fluorescence-quenching properties pose a challenge for biomolecular investigations that are based on direct optical readouts. Here, we employ few-layer hexagonal boron nitride (hBN) as a precisely tailorable fluorescence spacer between labelled lipid membranes and graphene substrates. By stacking high-quality hBN crystals in the 10–20 nm thickness range on monolayer graphene, we observe distance-dependent fluorescence intensity variations. Remarkably, with hBN spacers as thin as 20 nm, the fluorescence intensity is comparable to bare SiO₂/Si substrates, while the intensity was reduced to 60 % and 80 % with ~10 nm and ~16 nm hBN thicknesses respectively. We confirm that pre-determined hBN thicknesses can be employed to control the non-radiative energy transfer properties of graphene, with fluorescence quenching following a d⁻⁴ distance-dependent behaviour. This seamless integration of electronically active and dielectric van der Waals materials into vertical heterostructures enables multifunctional platforms addressing the manipulation, localization, and visualization of biomolecules for fundamental biophysics and biosensing applications.

Optical emitters in hexagonal boron nitride (hBN) are promising probes for single-molecule sensing platforms. When engineered in nanoparticle form, they can be integrated as detectors in nanodevices, yet positional control at the nanoscale is lacking. Here we demonstrate the functionalization of DNA origami nanopores with optically active hBN nanoparticles (NPs) with nanometer precision. The NPs are active under three wavelengths of visible illumination and display both stable and blinking emission, enabling their accurate localization by using wide-field optical nanoscopy. Correlative opto-structural characterization reveals deterministic binding of bright, multicolor hBN NPs at the pore rim due to π-π stacking interactions at site-specific locations on the DNA origami. Our work provides a scalable, bottom-up approach toward deterministic assembly of solid-state emitters on arbitrary structural elements based on DNA origami. Such a nanoscale arrangement of optically active components can advance the development of single-molecule platforms, including optical nanopores and nanochannel sensors.