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Probing Streptavidin-Biotin Interaction and Complex Structure at the Single-Molecule Level Using a Self-Crowded

Santosh Khatri1, Jin He1,2

  • 1Physics Department, Florida International University, Miami, Florida, USA.

Small (Weinheim an Der Bergstrasse, Germany)
|December 31, 2025
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Summary

This study enhances nanopipette sensing for label-free detection of small molecule interactions. The method reveals complex stoichiometry and structure with high spatial resolution, advancing biomolecular interaction analysis.

Keywords:
intermolecular interactionsmolecular crowdingnanopipettenanoporesingle‐molecule analysisstreptavidin‐biotin interaction

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Area of Science:

  • Biophysics
  • Analytical Chemistry
  • Molecular Biology

Background:

  • Probing biomolecular interactions is crucial for understanding biological systems and drug development.
  • Current methods face challenges in label-free, single-molecule detection of small molecules under physiological conditions.
  • Nanopipette-based nanopore sensing offers a cost-effective solution for label-free single-molecule analysis in solution.

Purpose of the Study:

  • To improve nanopipette sensitivity for detecting small molecule interactions.
  • To apply this enhanced method to study the stoichiometry and structure of molecular complexes.
  • To investigate the influence of external factors on molecular complex formation.

Main Methods:

  • Utilized a self-crowding technique to enhance nanopipette sensitivity.
  • Employed streptavidin-biotin interaction as a model system for studying molecular binding.
  • Analyzed unique multipeak ionic current spikes generated during molecular interactions.

Main Results:

  • Observed unique multipeak ionic current spikes indicating complex formation.
  • Demonstrated that peak characteristics correlate with biotin conjugate size, revealing stoichiometry and surface contour.
  • Showed that crowding levels and concentration ratios influence complex stoichiometry.

Conclusions:

  • The enhanced nanopipette approach provides high spatial resolution for characterizing label-free small molecule interactions.
  • This method is promising for studying molecular interactions in crowded environments.
  • The technique enables high-resolution structural characterization of molecular complexes.