A nanopore-gated sub-attoliter silicon nanocavity for non-invasive single molecule trapping and analysis

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 sub-attoliter silicon nanocavity that enables precise, non-invasive trapping of individual biomolecules for extended observation times, eliminating the need for surface immobilisation or external forces. Using nucleosomes as model systems, we demonstrate single-molecule Förster resonance energy transfer (smFRET) to monitor relative distances and directly observe dynamic unwrapping and rewrapping events induced by the chromatin remodelling enzyme Chd1. Our data further demonstrate that an applied electrical field can modulate the conformational properties of the macromolecules, emphasizing a key advantage of our device: it does not require an electrical field to retain trapped molecules. We envision this nanocavity platform as a powerful tool for the non-invasive interrogation of molecular dynamics in physiologically relevant environments, offering unperturbed access to weak and transient interactions that are central to biological regulation.