• phase imaging
• nanoindentation
• AFM
• protein swelling

AFM microscopy is a very promising tool for the understanding and the study of biological materials. This abstract briefly shows the results obtained during the period of my Ph.D. studies in the Chemistry and Industrial Chemistry Department at the University of Pisa. A commercial atomic force microscope (AFM) was used to investigate different kinds of biomaterials such as oligo peptides, polymers and proteins but also some hard materials as silicon and metals.
The AFM consists of a microsized cantilever with a sharp tip (probe) at its end that is used to scan the specimen surface. The cantilever is typically made of silicon or silicon nitride with a tip radius of curvature on the order of few nanometers. When the tip is brought into proximity of a sample surface, forces between the tip and the sample lead to a deflection of the cantilever ruled by Hooke's law. Depending on the situation, forces that are measured in AFM include mechanical contact force, Van der Waals forces, capillary forces, chemical bonding, electrostatic forces etc. Traditionally, the sample is mounted on a piezoelectric scanner, that can move the object under examination in the z direction for maintaining a constant force, and in the x and y directions for scanning the sample. An image of the surface is obtained by mechanically moving the probe in a raster scan (that is the pattern of image detection and reconstruction in a computer image) over the specimen, line by line, and recording the probe-surface interaction as a function of position.
The operating mode described above represents the typical way to use the atomic force microscope. But a whole world of capabilities of the instrument can be used. In particular we focused our attention on three research lines:
• The phase imaging
• The mechanical analysis of materials
• The chemical force microscopy
The AFM, developed first to explore atomic details on hard materials, has evolved to an imaging method capable of achieving fine structural details on biological samples and soft matter. The first one in fact, was used in order to characterize the shape and the morphology of particular bio samples: some oligopeptides that could auto aggregate on complex structures depending on the concentration of the starting solutions from which they are prepared and on the presence or not buffer salt. The measurements were performed in the so called “tapping mode” which is capable of acquiring both the morphological maps and also the phase maps. This signal is a powerful extension of AFM that provides nanometer-scale information about surface structure and properties often not revealed by traditional techniques. In phase imaging, the phase lag of the cantilever oscillation, relative to the drive signal, is simultaneously monitored with topography data. The phase lag is very sensitive to variations in many material properties such as viscoelastic properties and this allows for a precise determination of the presence of organic materials
What we have found is a dependence of peptide aggregates dimensions from the starting concentration. Essentially a growing trend is found with the augmentation of concentration regarding both the mean dimension and the dispersion of aggregates. Moreover a similar trend was found also in peptides prepared
from a salt solution. Nevertheless in this case the dispersion was quite minimal: the presence of the salt strongly influences the dimension of peptides structures. For a better understanding of the aggregation process it would be interesting, for future works, to monitor the dynamics of the peptide aggregations during the cast of the solvent and to make more measurements of samples from solution at different concentration.
The second argument we deal with was the mechanical analysis of materials. Tissues are a challenging class of materials as they are composed in hierarchical structures with important features down to the nanometer scale. Continuing developments in indentation data model and analysis will increase the usefulness of the method for the characterisation of biomaterials and in particular for tissue regeneration. The nanoindentation, also known as depth sensing indentation (DSI), involves the application of a controlled load over the surface to induce local deformations. Load and displacement are monitored during the loading- unloading curves enabling the calculation of the interested mechanical properties. Some theoretical models were considered and new ones were developed in order to get a better understanding of phenomena involved during the indentation process. A technique that can probe mechanical properties at these scales has the potential to answer numerous questions that are relevant in the field of nanotribology and nanomechanics. Several tests were performed over a large variety of materials including PMMA, polystyrene, silicon, metals and so on in order to obtain two of the most important mechanical properties: the hardness and the Young modulus. Moreover other deeper studies allow for the determination of the hardness in function of the indentation depth, the stiffness and other important features.
Anyway the results obtained have to be fully understood due the large variety of theories and method of analysis of the data. We have also to take in account the instrument data distortion and the different materials response to indentation tests that could affect the final results.
The last research addressed to biomaterials in this work is the chemical force microscope, exploited to monitor the forces involved in a protein swelling experiment. The potential of the AFM to reveal ultra low forces at high lateral resolution has opened an exciting way for measuring inter and intra molecular forces at the single molecule level.
In particular Human Serum Albumin was used for this test. The idea is to detect and study the binding of ligands on tips to surface-bound receptors by applying an increasing force to the complex that reduces its lifetime until it dissociates at a measurable unbinding force. During the loading unloading curves a couple of step (revealing the sudden change) have been found revealing the first a small detachment of the protein from the surface, while the second is properly due to the uncoiling of albumin.
Several measurements were collected in order to have statistically significant data.