Acoustic Force Spectroscopy

An introductory explanation of the working principle of acoustic force spectroscopy.

Acoustic Force Spectroscopy (AFS) is a single-molecule and single-cell manipulation technology that applies acoustic forces on multiple biomolecules and cells while tracking them in 3D with high accuracy.

This enables the measurement of the physical properties of hundreds of cells and molecules in real-time, allowing the user to receive high-throughput data within one experiment.

Working principle

The AFS technology consists of a glass microfluidic chip with a piezo element. An applied voltage drives this piezo element and resonantly excites planar acoustic waves (ultrasound) over a flow cell. These waves apply forces on micrometer-sized particles with a different density than the surrounding medium, such as polystyrene microspheres or cells. The acoustic force can be adjusted by tuning the driving voltage on the piezo element and is defined as:

Acoustic Force Spectroscopy Working Principle

where V is the volume of the particle, κ* (=κpm) is the compressibility ratio between the particle and the medium, respectively, ρ* (=ρpm) is the density ratio between the particle and the medium, respectively, and v is the acoustic velocity.

AFS and single-molecules

The figure shows a typical AFS experimental setup, where multiple molecules, such as DNA molecules are tethered between the surface of the chip and microspheres.

When turning on the acoustic forces, the microspheres experience a force along the vertical (z) direction of the standing wave. This results in the stretching of the DNA molecules towards the acoustic node.

By measuring the z-position of the beads, the extension and mechanical properties of the biomolecules can be determined.

The importance of measuring many biomolecules in parallel lies in the fact that many independent measurements are often needed to distinguish heterogeneous behavior and rare events from intrinsic stochastic behavior caused by thermal fluctuations.

When investigating the mechanical properties of biomolecular structures, such as DNA or proteins, it is essential to be able to apply a wide range of forces, so as to induce conformational changes such as protein unfolding or bond ruptures. AFS features a large force range from 0 to 200 pN, ensuring that these applications can be performed. In combination with the fast loading rates ranging from 10-4 pN/s to 103pN/s, it is possible to conduct these experiments in a massively parallel manner.

Working principle AFS

Force-distance measurements

Examples of typical data output using the AFS technology include force-distance curves of multiple single molecules, such as DNA, under different conditions (left figure). The tethered molecules can be promptly tested
in the presence of different molecules of varying concentrations or in different buffers.

AFS force-distance curves example

Constant force measurements

Equilibrium dynamics of biomolecular states can be measured by performing constant force measurements on many molecules in parallel.

AFS Equilibrium dynamics showing protein Unfolding example


  1. Sitters, G.; Kamsma, D.; Thalhammer, G.; Ritsch-Marte, M.; Peterman, E.J.G.; Wuite, G.J.L. (2015). Acoustic force spectroscopy. Nature Methods12: 47-50.
  2. Kamsma, D.; Creyghton, R.; Sitters, G.; Wuite, G.J.L.; Peterman, E.J.G. (2016). Tuning the Music: Acoustic Force Spectroscopy (AFS) 2.0. Methods105: 26-33.

Available Products

AFS™ Parallel Single-Molecule Force Spectroscopy

Parallel Single-Molecule Force Spectroscopy

Acoustic Force Spectrosocpy (AFS™) is a new single-molecule and single-cell manipulation method capable of applying acoustic forces on hundreds of biomolecules in parallel for precise experimentation with high throughput. It enables scientists to probe thousands of individual molecules in parallel (such as RNA, DNA, proteins, and living cells), allowing statistical analysis of the mechanical properties of biological properties of biological systems based on a single experiment.

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