AFS is the first fast and easy-to-use instrument for highly parallel single-molecule manipulation and measurement. The technology is based on a cost effective lab-on-chip device in which ultrasonic waves exert piconewton forces on thousands of biomolecules in parallel.
Acoustic Force Spectroscopy, or AFS? is a single-molecule, lab-on-a-chip technology in which acoustic waves (ultrasound) are employed to exert forces from sub-pN to hundreds of pNs on thousands of biomolecules (such as DNA, RNA or proteins) in parallel, with sub-millisecond response time and inherent stability.
AFS? represents an ideal tool to perform a wide variety of experiments and decipher the details of molecular interactions at the level of nucleic acids-proteins, drug-protein and antigen-antibody. AFS enables scientist to investigate structure-function of proteins, new biological mechanisms, compound screening and cell mechanics. In addition, free energy diagrams, kinetic rates and intermediate states in reaction pathways can also be studied and identified.
AFS? technology consists of a glass micro-fabricated microfluidic chip and includes transparent piezoelectric transducers that generate acoustic waves. The contact wires are directly deposited on the AFS? device and a robust and reliable electric and fluidic connection is insured using our custom developed AFS? chip-holder. AFS? is a versatile instrument to perform cutting-edge biological and biomedical research at the single-molecule level.
AFS? Stand-alone
The stand-alone AFS package includes an inverted microscope, microfluidics, workstation, software and electronics. This dedicated easy-to-use system allows turnkey operations and measurements.
AFS? Module
The AFS module is designed as a highly flexible instrument and can be easily implemented on any custom or commercial inverted microscope using straightforward bright field illumination. Implementing the AFS module on your own microscope set-up allows a wide variety of applications.
AFS? Applications
AFS? technology can be employed in numerous different applications. Here we demonstrate three singlemolecule measurements that demonstrate the precision and multiplexing capabilities of AFS?.
Applications include:
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Dynamic force spectroscopy
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Constant-force measurements
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Force-distance curves
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Biopolymer mechanics
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Bond Rupture
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Micro-rheology
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Cell mechanics
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Mechanical properties of hydrogels
High Precision Force and Length Measurement
AFS? is an ideal tool to perform high-precision force measurements on single biomolecules. Here we demonstrate the accuracy AFS? by applying various level of tension on a single DNA molecule and measure its length using the AFS? tracking software.
The experiment is performed on a single DNA molecule (8.4 kbps in length) tethered to a streptavidin coated polystyrene microsphere (4.5 μm in diameter).
Prior to the measurement a look-up-table is generated and the microsphere’s height can be tracked with nm-accuracy.
During the measurement we increase the amplitude of the driving voltage from 0 to 2.1 Volt Peak-to-Peak (Vpp), corresponding to an increase in the acoustic force from 0 to 15.8 pN. The AFS? software determines the DNA length at various forces in real-time to detect biological events, e.g.protein-DNA interactions.
The 14bit vertical resolution of the function generator allows generating constant acoustic forces with sub-pN precision, stability over hours and
sub-ms response time.
Measuring Mechanical Properties in Parallel
AFS? has the powerful and unique capability of performing singlemolecule measurements in a highly parallel fashion. This is of pivotal importance since often many independent measurements are needed to distinguish heterogeneous behavior and rare events from intrinsic stochasticity caused by thermal fluctuations. In this example we perform a single-molecule experiment in which we simultaneously apply tension and measure the mechanical properties of 20 DNA molecules in parallel.
Force - Extension Curve
Stretching curves of 20 individual DNA molecules in parallel. We apply increasing tensions from 0.1 to 48 pN to record the force-extension profiles. The curves are obtained in parallel, simultaneously and in a single field of view. The ability of multiplexing mechanical measurements at the single-molecule level can be readily applied to different assays such as protein and RNA unfolding and nucleic acids processing enzymes.
Highly Multiplexed Bond Rupture Measurement
To characterize the strength of antibody-antigen interactions statistical analysis and high data throughput is of crucial importance. Due to the massive multiplexing capabilities of AFS?, a large number of bonds can be probed in parallel, greatly reducing measurement times.
Here we show how AFS? is able to obtain a complete rupture-force distribution of the Dig::anti-Dig bond in a single measurement of 251 single-molecule measurement in parallel.
Rupture Force Histogram
251 individual Dig::anti-Dig bonds are stretched with a loading rate of 2.3 pN/s. The rupture force is of each Dig::anti-Dig bond is recorded and plotted into a rupture force histogram. This histogram was obtained in a single experiment under identical conditions, insuring experiment reliability and reproducibility. AFS? is able to apply loading rates that span several orders of magnitude (10-4 pN/s to 103 pN/s), making at an ideal tool for highly parallel dynamic force spectroscopy measurements.
AFS? Chips
Three individual AFS? chips are included with the AFS? system. Each AFS? chip is pre-calibrated by LUMICKS and the resonance frequencies are made known to the buyer.
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Range of loading rates 10-4 pN/s to 103 pN/s
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AFS? chip size (configurable) 45 mm x 15 mm x 1.275 mm
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Maximum force applicable >200 pN (4.5 μm polystyrene microspheres, using voltage amplifier)
AFS? Workstation & Software (upgradable)
AFS? includes a LabView2013 software package and a powerful workstation. The AFS? software is capable of real-time, nanometer resolution tracking of thousands of microspheres in parallel.
Key Features
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Real-time parallel 3D tracking, 60-300 microspheres at 25 Hz
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Real-time parallel 2D tracking, 300-1500 microspheres at 25 Hz
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Real-time position tracking accuracy (X,Y) of 2 nm at 25 Hz
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Real-time height tracking accuracy (Z) of 5 nm at 25 Hz
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Z-drift correction through reference bead differential tracking
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Automated look-up-table calibration
AFS? Amplifier (optional)
AFS? capabilities can be expanded by using a voltage amplifier. Amplifying the voltage increases the maximum force that can be applied on the biomolecules.
The amplifier can be easily connected to the AFS? via the back panel. All relevant software and drivers have been pre-installed on the AFS? computer to accommodate the voltage amplifier.
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