Stretching Single Collagen Fibrils Reveals Nonlinear Mechanical Behavior.

The mechanical properties of collagen fibrils play an important role in cell-matrix interactions and are a manifestation of their molecular structure. Using a, to our knowledge, novel combination of uniaxial, longitudinal straining and radial nanoindentation, we found that type I collagen fibrils show a pronounced nonlinear behavior in the form of strain stiffening at strains from 0 to 15%, followed by strain softening at strains from 15 to 25%. At the molecular scale, this surprising phenomenon can be explained by the combination of unfolding of disordered domains and breaking of native cross-links at different stages of strain. Fibrils cross-linked artificially by glutaraldehyde do not show such a behavior, and nanoindentation allowed us to measure the mechanics of the overlap and gap regions in the D-banding individually. The results could have consequences for our understanding of matrix mechanics and the influence of excessive glycation, which has been linked with age-related diseases such as diabetes. Furthermore, the simplicity of the straining method could be attractive in other areas of biophysics at the nanometer scale because it does not require any bespoke instrumentation and is easy to use.

Mechanical properties of collagen fibrils determined by buckling analysis.

The mechanical properties of biological nanofibers such as collagen fibrils are important in many applications, ranging from tissue-engineering to cancer treatment. However, mechanical testing is not straightforward at the nanometer scale. Here, we use the theory of column-buckling to determine the bending properties of individual collagen fibrils. To achieve this, fibrils were deposited on a manually pre-stretched foil, which was then released with the fibrils attached. Atomic Force Microscopy (AFM) imaging was used to determine the tensile modulus by measuring the buckling-wavelength and the radius for each fibril. Comparison with data obtained by AFM nanoindentation and other, more sophisticated methods, shows that our results are in very good agreement. The great advantage of this simple approach is that it can be used to quickly determine mechanical properties without force or stress-strain measurements, which are challenging to obtain accurately and at high throughput at the nanoscale. The method could be applied to any nanofibers, not just collagen fibrils. STATEMENT OF SIGNIFICANCE: Collagen fibrils are the main constituent of the extracellular matrix, and alterations of their mechanical properties can have significant effects on cell adhesion and motility. This has, ultimately, implications in age-related diseases and cancer. Furthermore, tuning the mechanical properties of collagen fibrils could be an important tool in the design of artificial cell scaffolds in tissue-engineering. For these reasons, it is important to have methods that can be used to determine the mechanical properties of fibrils at the single-fibril level and, therefore, at the nanometer scale. The method presented here has the advantage of being easy to use and avoids some of the fundamental issues of more established methods.

AC Kelvin Probe Force Microscopy Enables Charge Mapping in Water.

Mapping charged chemical groups at the solid-liquid interface is important in many areas, ranging from colloidal systems to biomolecular interactions. However, classical methods to measure surface charges either lack spatial resolution or─like Kelvin-probe force microscopy (KPFM)─cannot be applied in aqueous solutions because a DC bias voltage is used. Here, we show that using AC Kelvin probe force microscopy (AC-KPFM), in which the DC bias is replaced with an AC voltage of sufficiently high frequency, the surface potential of spatially fixated, charged surface groups can be mapped in aqueous solution. We demonstrate this with micropatterned, functionalized alkanethiol layers which expose ionized amino- and carboxy-groups. These groups are representative of the charged groups of most biomolecules such as proteins. By adjusting the pH of the solution, the charge of the groups was reversibly altered, demonstrating the electrostatic nature of the measured signal. The influence of the electric double layer (EDL) on the measurement is discussed, and we, furthermore, show how charged, micropatterned layers can be used to spatially direct the deposition of nanoparticles of opposite charge.

Mechanical Strain Alters the Surface Charge of Collagen Fibrils.

Collagen fibrils act like nanoscale cables in the extracellular matrix of vertebrate tissues and provide a scaffold for cells to attach to. However, beyond this mechanical function, the surface charge of collagen fibrils is also likely to play an important role. Here, we show that native, type I collagen fibrils from a mammal tendon exhibit a particular dependence of surface charge on longitudinal strain. Fibrils first become more positive with strain of up to 10% and then become more negative again with strain between 10 and 17%. The effect correlates with the stiffness of fibrils and can be explained by structural rearrangements, which expose hidden, ionizable residues. Fibrils treated with glutaraldehyde did not show any change in surface charge when strained. The electrical surface potential, which is directly related to the number ratio of exposed amine and carboxy groups on the surface, was determined by Kelvin-probe force microscopy of fibrils attached on an extensible, thin polymer film. By stretching the film, a large number of individual fibrils could be strained simultaneously without resorting to sophisticated nanomechanical devices. It is conceivable that cells react to such changes of the fibril charge and that this effect is an additional contributor, besides mechanics, to a number of physiological processes. It may also need to be considered in the design of tissue-engineering scaffolds.

Imaging surface charges of individual biomolecules.

Surface charges play a key role in determining the structure and function of proteins, DNA, and larger biomolecular structures. Here we report on the measurement of the electrostatic surface potential of individual DNA and avidin molecules with nanometer resolution using Kelvin probe force microscopy. We also show, for the first time, the surface potential of buffer salts shielding individual DNA molecules, which would not be possible with conventional ensemble techniques.

Glycation changes molecular organization and charge distribution in type I collagen fibrils.

Collagen fibrils are central to the molecular organization of the extracellular matrix (ECM) and to defining the cellular microenvironment. Glycation of collagen fibrils is known to impact on cell adhesion and migration in the context of cancer and in model studies, glycation of collagen molecules has been shown to affect the binding of other ECM components to collagen. Here we use TEM to show that ribose-5-phosphate (R5P) glycation of collagen fibrils - potentially important in the microenvironment of actively dividing cells, such as cancer cells - disrupts the longitudinal ordering of the molecules in collagen fibrils and, using KFM and FLiM, that R5P-glycated collagen fibrils have a more negative surface charge than unglycated fibrils. Altered molecular arrangement can be expected to impact on the accessibility of cell adhesion sites and altered fibril surface charge on the integrity of the extracellular matrix structure surrounding glycated collagen fibrils. Both effects are highly relevant for cell adhesion and migration within the tumour microenvironment.

Evaluation of surface charge shift of collagen fibrils exposed to glutaraldehyde.

Collagen fibrils are a major component of the extracellular matrix. They form nanometer-scale "cables" acting as a scaffold for cells in animal tissues and are widely used in tissue-engineering. Besides controlling their structure and mechanical properties, it is crucial to have information of their surface charge, as this affects how cells attach to the scaffold. Here, we employed Kelvin-probe Force Microscopy to determine the electrostatic surface potential at the single-fibril level and investigated how glutaraldehyde, a well-established protein cross-linking agent, shifts the surface charge to more negative values without disrupting the fibrils themselves. This shift can be interpreted as the result of the reaction between the carbonyl groups of glutaraldehyde and the amine groups of collagen. It reduces the overall density of positively charged amine groups on the collagen fibril surface and, ultimately, results in the observed negative shift of the surface potential measured. Reactions between carbonyl-containing compounds and proteins are considered the first step in glycation, the non-enzymatic reaction between sugars and proteins. It is conceivable that similar charge shifts happen in vivo caused by sugars, which could have serious implications on age-related diseases such as diabetes and which has been hypothesised for many years.

Guiding self-assembly with the tip of an atomic force microscope.

We report the guided self-assembly of nanoparticles to geometrically well-defined charge patterns written on a dielectric surface with the conductive tip of an atomic force microscope (AFM). Charges are deposited in 30-90-nm thick fluorocarbon layers by applying voltage pulses to the conductive AFM tip. The samples are being developed by dipping them into an organic suspension of silica nanoparticles. Coulomb forces draw the nanoparticles to the charge patterns. With this simple process, we achieve a resolution of about 800 nm.

Young's modulus measurement on pig trachea and bronchial airways.

Young's Modulus was measured on the trachea and first three generations of pig airways by compression. A simple and low-cost system for measuring the elastic properties of small bio-materials is presented. The force-displacement measurements have been undertaken on dissected cartilage and trachea mucosa from pig trachea and bronchial segments. Young's Modulus of trachea wall, 1.78 ± 0.51 MPa, is found to be dominated by the trachea cartilage of value 1.74 ± 0.85 MPa while the modulus for trachea mucosa was 0.15 ± 0.03 MPa. The Young's Modulus of the airway wall from the first three generations of bronchi decreases from 1.35 ± 0.17 to 0.35 ± 0.10 MPa which is also found to be dominated by the airway cartilage. Airway mucosa is found to have similar Young's modulus of 0.036 ± 0.005 MPa for the first three generations of bronchial airways.

Simultaneous investigation of the influence of topography and charge on protein adsorption using artificial nanopatterns.

The combined influence of surface topography and charge of a polymer surface on the adsorption of the protein avidin has been investigated. Atomic force microscopy contact mode imaging and charge writing were used to create defined topographical roughness and electrostatic charge patterns on the surface of polystyrene. Increased avidin adsorption was found on nanometer-size topographical patterns, but the adsorption remained unaffected by electrostatic patterns.

Mechanical properties of collagen fibrils.

The formation of collagen fibers from staggered subfibrils still lacks a universally accepted model. Determining the mechanical properties of single collagen fibrils (diameter 50-200 nm) provides new insights into collagen structure. In this work, the reduced modulus of collagen was measured by nanoindentation using atomic force microscopy. For individual type 1 collagen fibrils from rat tail, the modulus was found to be in the range from 5 GPa to 11.5 GPa (in air and at room temperature). The hypothesis that collagen anisotropy is due to the subfibrils being aligned along the fibril axis is supported by nonuniform surface imprints performed by high load nanoindentation.

Morphology and mechanical stability of amyloid-like peptide fibrils.

Synthetic, amyloid-like peptide fibrils have recently attracted interest as a novel, potentially biocompatible material for applications in biotechnology and tissue-engineering. In this paper, we report atomic force microscopy (AFM) studies of the morphology and mechanical stability of fibrils self-assembled in vitro from the short peptide TTR(105-115), which serves as a model system for amyloid fibrils. It forms predominantly straight rods of approximately 1 microm in length and of diameters between 7 nm and 12 nm. We found polymorphism, with some fibrils exhibiting an unstructured morphology and others showing a regular, longitudinal surface pattern of 90 nm periodicity. Contact mode AFM-imaging in air was utilised to perform mechanical tests of individual fibrils on the nanometer scale with a defined, vertical force in the nN-range applied by the AFM-tip. Above 100 nN, all fibrils showed a permanent, mechanical deformation whereas below 40 nN, fibrils remained unaffected. Tapping-mode AFM-imaging in water led to fibril decomposition within 1.5 h whereas tapping-mode imaging in air left fibrils intact. Additional investigations by circular-dichroism spectroscopy showed that dispersed fibrils were structurally stable in aqueous solution between pH 3 and pH 8, and in sodium phosphate buffer of concentration between 50 mM and 1 M.

Preparation and characterization of dense films of poly(amidoamine) dendrimers on indium tin oxide.

Indium tin oxide (ITO) substrates were modified with a layer of poly(amidoamine) (PAMAM) dendrimers to change their surface properties and, in particular, the substrates' work function. The functionalization procedure involved the electrostatic adsorption of positively charged PAMAM dendrimers of generation five onto negatively polarized ITO surfaces. Three different PAMAM dendrimers were used: PAMAM-NH2 and PAMAM-OH with terminal amine and hydroxyl groups, respectively, as well as Q-PAMAM-NH2, which had been prepared from PAMAM-NH2 by quaternization of the dendrimer's terminal and internal amine groups with methyl iodide. The resulting organic films were analyzed by contact angle goniometry, X-ray photoelectron spectroscopy, ellipsometry, and Kelvin probe force microscopy to confirm the presence of a dense layer. A Langmuir isotherm was derived from surface densities of fluorescence-labeled PAMAM-NH2 dendrimers from which we deduced an equilibrium binding constant, K(eq), of (1.3 +/- 0.3) x 10(5) M(-1). Kelvin probe measurements of the contact potential difference revealed a high reduction of the work function from 4.9 eV for bare ITO to 4.3 eV for ITO with a dense film of PAMAM-NH2 of generation five. PAMAM-OH and Q-PAMAM-NH2 resulted in slightly smaller work function changes. This study illustrates that the work function of ITO can be tuned by adlayers composed of PAMAM dendrimers.

Patterning amyloid peptide fibrils by AFM charge writing.

Surface charge patterns generated by atomic force microscopy-based charge writing were used to pattern amyloid-like peptide fibrils on a solid substrate. Fibrils of the short peptide TTR105-115 were encapsulated inside water droplets of a water-in-perfluorocarbon oil emulsion and retained their rod morphology. They were observed to deposit selectively with a lateral resolution of approximately 1 microm onto negatively charged patterns on a polymethyl-methacrylate substrate.