An introductory explanation of the working principle of acoustic force spectroscopy.
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.
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:
where V is the volume of the particle, κ* (=κp/κm) is the compressibility ratio between the particle and the medium, respectively, ρ* (=ρp/ρm) 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.
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.
Constant force measurements
Equilibrium dynamics of biomolecular states can be measured by performing constant force measurements on many molecules in parallel.
With AFS™ scientists are able to probe, manipulate and measure thousands of single molecules or single cells in parallel. The AFS™ provides precise experimentation with high throughput, allowing statistical analysis of the mechanical properties of biological systems based on a single experiment.
- Sitters, G.; Kamsma, D.; Thalhammer, G.; Ritsch-Marte, M.; Peterman, E.J.G.; Wuite, G.J.L. (2015). Acoustic force spectroscopy. Nature Methods. 12: 47-50.
- Kamsma, D.; Creyghton, R.; Sitters, G.; Wuite, G.J.L.; Peterman, E.J.G. (2016). Tuning the Music: Acoustic Force Spectroscopy (AFS) 2.0. Methods. 105: 26-33.