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Advancing Single-Molecule Nanopore Sensing: From Protein Conformation and Metal Binding to DNA Transport via Hybrid Nanostructures

Year: 2025
Type: article
Abstract: This dissertation presents the development and application of solid-state and hybrid nanopore platforms for single-molecule analysis of biomolecular interactions. First, a silicon nitride nanopore was employed to characterize the conformational states of the transferrin receptor (TfR), enabling discrimination between its monomeric and dimeric forms, as well as from its ligand, human serum transferrin (hSTf), despite their similar molecular weights. In a separate study, the same nanopore configuration was used to track the real-time binding of titanium ions to transferrin, demonstrating single-molecule sensitivity to metal-induced conformational changes. A hybrid microtubule–solid-state nanopore (MT–SSN) was then engineered, repurposing microtubules as electrostatically anchored conduits for ionic current. This hybrid system exhibited enhanced signal-to-noise ratios and significantly prolonged DNA translocation times, overcoming key limitations of traditional solid-state nanopores. Additionally, it provides the first experimental method to directly measure the electronic current through the microtubule lumen. Finally, a machine learning pipeline combining Gaussian mixture models and support vector machines was used to classify complex nanopore events and quantify the dissociation constants of SARS-CoV-2 Spike protein binding to candidate alternative receptors. This analysis yielded KD values of 17.4 ± 2.9 nM for Kremen1 and 126 ± 24 nM for ASGR1, closely aligning with ensemble flow cytometry data while offering a label-free, single-molecule alternative. Together, these contributions advance the precision and functionality of nanopore sensing, offering new tools for probing protein–protein interactions, metal coordination, nucleic acid transport, and viral receptor binding at the single-molecule level.
Language: English
Open Access status: green
OpenAlex ID: W7082113496
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