Probing Streptavidin-Biotin Interaction and Complex Structure at the Single-Molecule Level Using a Self-Crowded Nanopipette
Article
Khatri, Santosh, He, Jin. (2025). Probing Streptavidin-Biotin Interaction and Complex Structure at the Single-Molecule Level Using a Self-Crowded Nanopipette
. Small, e08579. 10.1002/smll.202508579
Khatri, Santosh, He, Jin. (2025). Probing Streptavidin-Biotin Interaction and Complex Structure at the Single-Molecule Level Using a Self-Crowded Nanopipette
. Small, e08579. 10.1002/smll.202508579
Probing intermolecular interactions between biomolecules (such as proteins and nucleic acids) or bioactive molecules (such as drug molecules) are important for both fundamental studies of living systems and various applications. Challenges remain for current methods in probing label-free small (bio)molecules involved interactions at the single-molecule level under physiological conditions. Nanopipette based nanopore sensing offers a powerful and cost-effective approach for label-free single-molecule detection and analysis in solution. In a recent study, we have greatly improved the sensitivity of nanopipettes for the detection of small molecules when the tip is self-crowded with molecules. Here, we applied this approach to study strong interactions involving small molecules, using streptavidin-biotin interaction as a model system. After mixing streptavidin and biotin, unique multipeak ionic current spikes, containing one large peak and up to four small peaks, appeared. The relative peak height of these small peaks increased when larger biotin conjugates were used. Therefore, these multipeak current spikes reveal the stoichiometry and surface contour of the formed complex, highlighting the extremely high spatial resolution of our method. We further investigated how external factors affect the stoichiometry of the complex. The average number of small peaks within each multipeak spike increases with both higher crowding levels at the nanopipette tip (induced by greater applied voltage) and higher biotin-to-streptavidin concentration ratios in the premixed solution. This study demonstrates the promising potential of this nanopipette approach for investigating interactions involving label-free small (bio)molecules in crowded environments and for high-resolution structural characterization of the resulting complexes.
