Tracking single-molecule ferritin reassembly and disassembly using polymer-coated nanopores

Ferritin is a multimeric protein that stores and releases iron, emerging as a promising candidate for nanomedicine, particularly in targeted drug delivery. Its ability to disassemble and reassemble under specific environments is crucial for encapsulating and releasing therapeutic payloads. In this work, we monitor ferritin reassembly and disassembly in real time at the single-molecule level using polymer-coated solid-state nanopores. The coating enabled continuous ion current recording of ferritin fragment translocations for up to 1 hour without clogging, overcoming a major challenge of uncoated nanopores. This long-term recording allowed us to track the full reassembly and disassembly processes. We show that while traditional population-based analysis can identify the presence of fragment mixtures during both reassembly and disassembly processes, it is unable to discriminate individual subunits due to heterogeneity in ferritin fragment mixtures. In contrast, using individual event analysis, we determined the volume and shape of individual fragments using single translocation events, enabling the identification of intermediate subunits (e.g., 4-, 6-, 8-, 10-,12-, and 16-mers) during the reassembly and disassembly processes. Through real-time tracking of ferritin reassembly and disassembly dynamics, this approach - combining nanopore coating and individual event analysis - demonstrates potential to characterize single proteins within a mixture, determine their compositions, and track reaction processes in real time.