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

• Published in: Small (Weinheim an der Bergstrasse, Germany) Vol. 19; no. 14; pp. e2206713 - n/a
• Main Authors: Kerkhoff, Yannic, Azizi, Latifeh, Mykuliak, Vasyl V., Hytönen, Vesa P., Block, Stephan
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 01.04.2023
• Subjects: Beads; Biotin; Cluster analysis; Fluorescence; high‐throughput measurements; Mechanical analysis; Microfluidics; Nanotechnology; Polyethylene glycol; Sensitivity; Shear forces; single‐molecule force spectroscopy; Spatial resolution; Spectrum analysis; total internal reflection fluorescence (TIRF) microscopy; total internal reflection fluorescence (TIRF) microscopy; high-throughput measurements; single-molecule force spectroscopy; microfluidics
• ISSN: 1613-6810; 1613-6829
• DOI: 10.1002/smll.202206713

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. A highly sensitive single‐molecule force spectroscopy method with high parallelization capabilities is introduced. The method uses a combination of microfluidics, total internal reflection fluorescence microscopy, and single‐particle tracking to quantify the nanomechanical properties of thousands of individual targets (macromolecules or ligand–receptor interactions), which are subject to well‐defined hydrodynamic shear forces (ranging between 0.03 and 20 pN).