A Nanopore-Gated Subattoliter Silicon Nanocavity for Single-Molecule Trapping and Analysis without Applying an External Force.

Biomolecules exhibit dynamic conformations critical to their functions, yet observing these processes at the single-molecule level under native conditions remains a formidable challenge. While surface immobilization has been widely used to extend observation times, it can disrupt molecular dynamics and impede biological function. Recent advancements in single-molecule trapping techniques have addressed some limitations, but achieving precise, controllable, long-term trapping in a molecularly crowded environment without external forces remains difficult. Here, we introduce a nanopore-gated subattoliter silicon nanocavity that enables precise entropic trapping of individual biomolecules for extended observation times, eliminating the need for surface immobilization or external forces. Using nucleosomes as model systems, we demonstrate single-molecule Förster resonance energy transfer (smFRET) to monitor relative distances. With smFRET, we directly observe dynamic unwrapping and rewrapping events induced by the chromatin remodeling enzyme Chd1, as well as weak interactions between two nucleosomes trapped inside the nanocavity. Our data further demonstrate that an applied electric field can modulate the conformational properties of the macromolecules, emphasizing a key advantage of our device: it does not require an electric field to retain trapped molecules. We envision this nanocavity platform as a powerful tool for the interrogation of molecular dynamics without applying an external force in physiologically relevant environments, offering access to weak and transient interactions that are central to biological regulation.