By AZoNano.com Staff Writers
Experimental Procedure and Results
The physiological functions of biological samples are often closely linked to their mechanical properties. This leads to a new field called mechanobiology. Moreover, a trend from qualitative analysis of organism as whole to quantitative testing of their smallest unit, for instance, the single cell, can be observed in many different biological research fields. This causes an increased demand for high precision microforce sensing equipment which facilitates quantitative micromechanical investigation of biological samples immersed in liquids.
The functionalities of proteins can be better understood through the measurement of their mechanical properties. For instance, silk fibers from web-spinning spiders exhibit optimal performance in air. They can extend as high as 3.7 times more than their length at zero strain and demonstrate a contraction with significant hysteresis, which enables spiders to catch their prey. Fibronectin (Fn) fibers are protein fibers present in extra cellular matrix of cells and connective tissue. They are vital in early wound repair. This article discusses the mechanical characterization of Fn fibers to gain insights into their structure and the mechanisms behind their folding and unfolding.
While performing the micromechanical investigation of samples immersed in liquids, the diffraction of the air-liquid interface makes it difficult to observe the sample from above. This can be addressed by using a combination of a FT-FS1000 Mechanical Probe and a high-resolution, inverted microscope, as depicted in Figure 1. This combination enables the quantitative micromechanical analysis of the liquid submerged Fn fiber.
Figure 1. FT-FS1000 Mechanical Probe on an inverted microscope
A FT-S1000-LAT Lateral Microforce Sensing Probe with a customized probe tip (attachment of a sharp tungsten probe), as shown in Figure 2, is employed to obtain the stress vs. strain curves and the young’s modulus of Fn fibers.
Figure 2. FT-S1000-LAT Lateral Microforce Sensing Probe
Experimental Procedure and Results
The first step is the deposition of Fn fibers across the trenches of a microfabricated PDMS grid, followed by their rehydration in PBS buffer. The sensing probe is then aligned corresponding to the Fn fiber and automated tensile tests of the fibers are performed using the FT-WFS02 Micromechanical Testing Software.
Figure 3. Relaxed single Fn fiber with a diameter of 3.5 ± 0.2 µm suspended over a trench with a width of 30 µm.
Figure 4. Stretched single Fn fiber
The tensile force applied to strain the Fn fiber, the measured stress (force per unit cross sectional area of the fiber), and the stiffness (slope of the stress-versus-strain curve) are described as functions of the measured fiber extension for one representative fiber, as illustrated in Figures 5 and 6.
The cross-sectional area of the Fn fibers is determined by optically measuring their diameters at zero strain. The assumption made is that the fibers maintain a uniform, circular diameter and constant volume when they are subjected to stretching.
Figure 5. Stress-versus-strain curve of a single Fn fiber
The stiffness of a single Fn fiber, as depicted in Figure 6, is not stable as anticipated for linearly elastic materials. On the other hand, the stress-versus-strain curves are largely nonlinear, being soft (compliant) at low extensions, and becoming rigid at high extensions.
There is a small increase in stiffness in the low strain region, but a drastic increase is witnessed when the fiber is subjected to beyond 150% strain. The change in the stiffness of a single fiber is in the orders of magnitude, from below 100 kPa to several MPa, corresponding to their relaxed condition to their highly stretched condition.
Figure 6. Young’s modulus as a function of the fiber elongation of a single Fn fiber
The fiber contraction and module refolding take place subsequent the release of the tensile force. Hence, measurements are taken to analyze the elasticity of the fibers and the rate at which they revert back to their original lengths after the release of the stress. Many materials exhibit plastic deformation before failure and such deformations result in irreversible slippage of molecules with one another.
The fibers’ contour length was measured several times subsequent to their release from the tip of the sensing probe. Immediately after their release, the fibers have exhibited a sinuous appearance and are contracted back to their initial length after a recovery period of a few minutes, as depicted in Figures 7a through 7d. All fibers analyzed have attained their original lengths as time progresses, irrespective of the state of extension.
Figure 7. a-d: Photograph of a Fn fiber during its recovery from the extended state
By reestablishing the force versus strain curves of the same fibers after a variable waiting period between each mechanical analysis, the status of recovery of the mechanical properties of the fibers is determined, as illustrated in Figures 8 and 9.
Figure 8. Results from the investigation of the mechanical property recovery after a short recovery period tw < 1 min
Figure 9. Results from the investigation of the mechanical property recovery after a long recovery period tw > 1 min
The force vs. strain curves exhibit substantial hysteresis when the waiting period between successive pulls is below 1 min. This implies that the second fiber extension needs only very low force to achieve a given strain corresponding to the first pull. This has been experienced by both initially relaxed and prestrained fibers.
Immediately after the onset of their contraction, the fibers are initially more compliant when compared to the original fibers. Nevertheless, they recover their initial mechanical properties after a waiting period of 1 min or more.
This test is highly significant considering the fact that the mechanical unfolding of Fn modules needs breaking of clusters of force-bearing backbone hydrogen bonds. The fibers’ ability to completely recover their mechanical strength reveals that these crucial hydrogen bonds have the ability to reform.
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This information has been sourced, reviewed and adapted from materials provided by FemtoTools.
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