Microfluidics-Based Force Spectroscopy Enables High-Throughput Force Experiments with Sub-Nanometer Resolution and Sub-Piconewton Sensitivity

Yannic Kerkhoff, Latifeh Azizi, Vasyl V. Mykuliak, Vesa P. Hytönen, Stephan Block

Research output: Contribution to journalArticleScientificpeer-review

1 Citation (Scopus)
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Several techniques have been established to quantify the mechanicals of single molecules. However, most of them show only limited capabilities of parallelizing the measurement by performing many individual measurements simultaneously. Herein, a microfluidics-based single-molecule force spectroscopy method, which achieves sub-nanometer spatial resolution and sub-piconewton sensitivity and is capable of simultaneously quantifying hundreds of single-molecule targets in parallel, is presented. It relies on a combination of total internal reflection microscopy and microfluidics, in which monodisperse fluorescent beads are immobilized on the bottom of a microfluidic channel by macromolecular linkers. Application of a flow generates a well-defined shear force acting on the beads, whereas the nanomechanical linker response is quantified based on the force-induced displacement of individual beads. To handle the high amount of data generated, a cluster analysis which is capable of a semi-automatic identification of measurement artifacts and molecular populations is implemented. The method is validated by probing the mechanical response polyethylene glycol linkers and binding strength of biotin–NeutrAvidin complexes. Two energy barriers (at 3 and 5.7 Å, respectively) in the biotin–NeutrAvidin interaction are resolved and the unfolding behavior of talin's rod domain R3 in the force range between 1 to ≈10 pN is probed.

Original languageEnglish
Article number2206713
Issue number14
Early online date2023
Publication statusPublished - 2023
Publication typeA1 Journal article-refereed


  • high-throughput measurements
  • microfluidics
  • single-molecule force spectroscopy
  • total internal reflection fluorescence (TIRF) microscopy

Publication forum classification

  • Publication forum level 3

ASJC Scopus subject areas

  • Biotechnology
  • General Chemistry
  • Biomaterials
  • General Materials Science


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