Characterization of Biomaterials by Atomic Force Microscopy

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

Responsive nanostructures for controlled alteration of interfacial properties

Responsive materials are a class of materials that are capable of “intelligently” changing properties upon exposure to a stimulus. Silk ionomers are introduced as a promising candidate of biopolymers that combine the robust, biocompatible properties of silk fibroin with the responsive properties of poly-l-lysine (PL) and poly-l-glutamic acid (PG). These polypeptides can be assembled using the well-known technique of layer-by-layer processing, allowing for the creation of finely tuned nanoscale multilayers coatings, but their properties remain largely unexplored in the literature. Thus, this research explores the properties of silk ionomer multilayers assembled in different geometries, ranging from planar films to three-dimensional microcapsules with the goal of created responsive systems. These silk ionomers are composed of a silk fibroin backbone with a variable degree of grafting with PG (for anionic species) or PL or PL-block- polyethylene glycol (PEG) (for cationic species). Initially, this research is focused on fundamental properties of the silk ionomer multilayer assemblies, such as stiffness, adhesion, and shearing properties. Elastic modulus of the materials is considered to be one of the most important mechanical parameters, but measurements of stiffness for nanoscale films can be challenging. Thus, we studied the applicability of various contact mechanics models to describe the relationship between force distance curves obtained by atomic force microscopy and the stiffness of various polymeric materials. Beyond considerations of tip size, we also examine the critical regions at which various commonly used indenter geometries are valid. Following this, we employed standard AFM probes and colloidal probes coated with covalently bonded silk ionomers to examine the friction and adhesion between silk ionomers layers. This technique allowed us to compare the interactions between silk ionomers of different chemical composition by using multilayer films containing standard silk ionomers or silk ionomers grafted with polyethylene glycol PEG. This led to the unexpected result that the PEG grafted silk ionomers experienced a higher degree of adhesion and a larger friction coefficient compared to the standard silk ionomers. Next, we move to microscale responsive systems based on silk ionomer multilayers. The first of these studies looks at the effect of assembly pH and chemical composition on the ultimate properties of hollow, spherical microcapsules. This study shows that all compositions and processing conditions yield microcapsules that show a substantial change in elastic modulus, swelling, and permeability, with maximum changes in property values (from acidic pH to basic pH) of around a factor of 6, 1.5, and 5, respectively. In addition, it was discovered that the use of acidic pH assembly inverts the permeability response (i.e. causes a drastic reduction in permeability at higher pH), whilst the use of PEG largely damps any observable trend in permeability, without adversely affecting the swelling or elastic modulus responses. In the second part of these studies, we constructed tri-component photopatterned arrays for the purpose of creating self-rolling films. This study demonstrated that the ultimate geometry of the final rolled shape can be tuned by controlling the thickness of various components, due to the creation of a stress mismatch at high pH conditions. Additionally, it was revealed that pH-driven, semi-reversible delamination of silk ionomers from polystyrene exhibited a change in both magnitude and wavelength with the addition of methanol treated silk fibroin as a top layer. Finally, we showcase examples of biologically compatible systems that incorporate non-polymeric materials in order to generate tunable optical behavior. In one study, we fabricated composite nanocellulose-silk fibroin meshes that contained genetically engineered bacteria that acted as chemically sensitive elements with a fluorescent response. The addition of silk fibroin was found to drastically improve the mechanical properties of the cellulose composite structures, safely contain the bacteria to prevent efflux into the medium, and protect the cells from moderate ultraviolet radiation exposure. The final study concludes with the creation of a self-assembled segmented gold-nickel nanorod array used as a responsive element when anchored into a hydrogen-bonded polymer multilayer. Because of the mild tethering conditions and the magnetic nickel component, the nanorods were able to tilt in response to an external magnetic field. This, in turn, allowed for the creation of a never before reported magnetic-plasmonic system capable of continuously-shifting multiple surface polariton scattering peaks (up to 100 nm shifts) with nearly complete reversibility and rapid (<1 s) response times. Overall, this research develops the understanding of the fundamental properties of several different species of silk ionomers and related polymeric materials. This understanding is then utilized to fabricate pH-responsive systems with drastic changes in modulus, permeability, and geometry. In the end, the research prototypes two types of systems with optical responses and chemical/magnetic stimuli, using materials that are chemically (i.e. silk fibroin-based) or structurally (i.e. multilayers) translatable to future work on silk ionomers. These projects all serve the purpose of advancing the understanding of materials and assembly strategies that will allow for the next generation of bioinspired responsive materials.Ph.D

Mechanical adhesion of SIO2 thin film on a polymeric substrate under compressive stress

International audienceTo ensure good adhesion between a 200 nm silicon dioxide layer and a 4.5 μm thick hardcoat polymeric coating, a better understanding of mechanisms of adhesion at this interface is needed. To reach this purpose, quantification of adhesion is performed by analyzing SiO2 buckle morphologies generated under compressive stress. This adhesion test was chosen for its representativeness of defects observed in real life. Interfacial toughness can be determined by applying Hutchinson & Suo model. This analytical model involves accurate value of elastic modulus Ef of SiO2 thin film. Small dimensions at stake make characterization of elastic modulus challenging. First part of the study focuses on using both nano-indentation and AFM to attempt assessment of SiO2 thin film elastic modulus. Results showed significant influence of substrate for both techniques. Impact on mechanical properties between SiO2 thin films with different intrinsic stresses was also investigated and suggests that higher density of SiO2 thin film leads to higher elastic modulus. Compression tests resulted in formation of straight-sided buckles that evolve into telephone cords upon unloading. Numerical simulation and Digital Image Correlation were implemented to ensure homogeneous strain of substrate and favor regular distribution of buckles. Values of energy release rates of SiO2 / Hardcoat range from 2.7 J/m² to 8.9 J/m², depending on moduli values found on wafer or lens substrate

Effects of mechanical properties on the reliability of Cu/low-k metallization systems

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 2007.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Includes bibliographical references (leaves 211-217).Cu and low-dielectric-constant (k) metallization schemes are critical for improved performance of integrated circuits. However, low elastic moduli, a characteristic of the low-k materials, lead to significant reliability degradation in Cu-interconnects. A thorough understanding of the effects of mechanical properties on electromigration induced failures is required for accurate reliability assessments. During electromigration inside Cu-interconnects, a change in atomic concentration correlates with a change in stress through the effective bulk modulus of the materials system, B, which decreases as the moduli of low-k materials used as inter-level dielectrics (ILDs) decrease. This property is at the core of discussions on electromigration-induced failures by all mechanisms. B is computed using finite element modeling analyses, using experimentally determined mechanical properties of the individual constituents. Characterization techniques include nanoindentation, cantilever deflection, and pressurized membrane deflection for elastic properties measurements, and chevron-notched double-cantilever pull structures for adhesion measurements. The dominant diffusion path in Cu-interconnects is the interface between Cu and the capping layer, which is currently a Si3N4-based film. We performed experiments on Cu-interconnect segments to investigate the kinetics of electromigration. A steady resistance increase over time prior to open-circuit failure, a result of void growth, correlates with the electromigration drift velocity. Diffusive measurements made in this fashion are more fundamental than lifetime measurements alone, and correlate with the combined effects of the electron wind and the back stress forces during electromigration induced void growth.(cont.)Using this method, the electromigration activation energy was determined to be 0.80±0.06eV. We conducted experiments using Cu-interconnects with different lengths to study line length effects. Although a reliability improvement is observed as the segment length decreases, there is no deterministic current-density line-length product, jL, for which all segments are immortal. This is because small, slit-like voids forming directly below vias will cause open-failures in Cu-interconnects. Therefore, the probabilistic jLcrit values obtained from via-above type nterconnects approximate the thresholds for void nucleation. The fact that jLcrit,nuc monotonically decreases with B results from an energy balance between the strain energy released and surface energy cost for void nucleation and the critical stress required for void nucleation is proportional to B. We also performed electromigration experiments using Cu/low-k interconnect trees to investigate the effects of active atomic sinks and reservoirs on interconnect reliability. In all cases, failures were due to void growth. Kinetic parameters were extracted to be ... Quantitative analysis demonstrates that the reliability of the failing segments is modulated by the evolution of stress in the whole interconnect tree. During this process, not only the diffusive parameters but also B play critical roles. However, as B decreases, the positive effects of reservoirs on reliability are diminished, while the negative effects of sinks on reliability are amplified.(cont.) Through comprehensive failure analyses, we also successfully identified the mechanism of electromigration-induced extrusions in Cu/low-k interconnects to be nearmode-I interfacial fracture between the Si3N4-based capping layer and the metallization/ILD layer below. The critical stress required for extrusion is found to depend not only on B but also on the layout and dimensions of the interconnects. As B decreases, sparsely packed, wide interconnects are most prone to extrusion-induced failures. Altogether, this research accounts for the effects of mechanical properties on all mechanisms of failure due to electromigration. The results provide an improved experimental basis for accurate circuit-level, layout-specific reliability assessments.by Frank LiLi Wei.Ph.D

EXTREME DYNAMICS OF NANOMATERIALS UNDER HIGH-RATE MECHANICAL STIMULI

Nanomaterials demonstrate novel mechanical properties attributed to the extremely large interfacial area. At quasi-static rates, the interfacial interactions are crucial in mechanical behaviors, however, materials under extreme mechanical stimuli are rarely studied at nanoscale. With an advanced laser-induced projectile impact test, we perform supersonic impact of micro-projectiles on polymer films, multilayer graphene, carbon- based nanocomposites membranes as well as individual micro-fibers, to study the interface interactions in the high-rate regime, and develop a simplified model to characterize the ballistic performance of materials

Nanoindentation testing of soft polymers : computation, experiments and parameters identification

Since nanoindentation technique is able to measure the mechanical properties of extremely thin layers and small volumes with high resolution, it also became one of the important testing techniques for thin polymer layers and coatings. This dissertation is focusing on the characterization of polymers using nanoindentation, which is dealt with numerical computation, experiments and parameters identification. An analysis procedure is developed with the FEM based inverse method to evaluate the hyperelasticity and time-dependent properties. This procedure is firstly verified with a parameters re-identification concept. An important issue in this dissertation is to take the error contributions in real nanoindentation experiments into account. Therefore, the effects of surface roughness, adhesion force, friction and the real shape of the tip are involved in the numerical model to minimize the systematic error between the experimental responses and the numerical predictions. The effects are quantified as functions or models with corresponding parameters to be identified. Finally, data from uniaxial or biaxial tensile tests and macroindentation tests are taken into account. The comparison of these different loading situations provides a validation of the proposed material model and a deep insight into nanoindentation of polymers.Da Nanoindentation die Messung der mechanischen Eigenschaften von dünnen Schichten und kleinen Volumen mit hoher Auflösung ermöglicht, hat sich diese Messmethode zu einer der wichtigsten Testmethoden für dünne Polymerschichten und -beschichtungen entwickelt. Diese Dissertation konzentriert sich auf die Charakterisierung von Polymeren mittels Nanoindentation, die in Form von numerischen Berechnungen, Experimenten und Parameteridentifikationen behandelt wird. Es wurde ein Auswertungsverfahren mit einer FEM basierten inversen Methode zur Berechnung der Hyperelastizität und der zeitabhängigen Eigenschaften entwickelt. Dieses Verfahren wird zunächst mit einem Konzept der Parameter Re-Identifikation verifiziert. Fehlerquellen wie Oberflächenrauheit, Adhäsionskräfte, Reibung und die tatsächlichen Form der Indenterspitze werden in das numerische Modell eingebunden, um die Abweichungen der numerischen Vorhersagen von den experimentellen Ergebnissen zu minimieren. Diese Einflüsse werden als Funktionen oder Modelle mit dazugehörigen, zu identifizierenden Parametern, quantifiziert. Abschließend werden Messwerte aus uni- oder biaxialen Zugversuchen und Makroindentationsversuchen betrachtet. Der Vergleich dieser verschiedenen Belastungszustände liefert eine Bestätigung des vorgeschlagenen Materialmodells und verschafft einen tieferen Einblick in die bei der Nanoindentation von Polymeren ablaufenden Mechanismen

Microstructural Effects on Diffusion and Mechanical Properties in Different Material Systems

Material microstructures is a very broad subject that encompasses most of the field of materials science. Advances in materials characterization and small scale mechanical experiments have brought about progress in the understanding of microstructural features and mechanisms down to the nanometer scale. In contrast to bulk features and properties, the small length scale of these microstructures lead to many interesting properties, and often requires a material-by-material, and even localized region-by-region study. While a thorough understanding of microstructural effects even in one material system is way beyond the scope of this thesis, there are nonetheless many common themes and properties that link together microstructures and their effects on different materials, especially in terms of mechanical properties. In this thesis, the effects of microstructural features such as grain boundaries, surface modification and structural hierarchy are investigated using two sample material systems: Cu-In-Ga-Se (CIGS) thin films and marine diatom frustules. We find that grain structures (or a lack there of) play a major role in both systems, and lead to differences in material stiffness, strength, and diffusion of species. The latter is also significantly affected by material defects across length scales, exemplified in CIGS by both microscopic voids and pores, and atomic scale like substitutional point defects. On the other hand, in diatoms, a low flaw density combined with an effective hierarchical design can propel the mechanical property of relatively simple ingredients like amorphous silica, to achieve extraordinary mechanical strength. We will conclude by showcasing that we can generalize some of these knowledge on microstructural effects across material systems, to help designing manmade structures that fully capture the material-level and structural-level properties of natural marine diatoms.</p

INVESTIGATION OF THE MECHANICAL PROPERTIES OF POLY (ETHYLENE GLYCOL) DIACRYLATE BY NANOINDENTATION USING ATOMIC FORCE MICROSCOPY

Poly (ethylene glycol) (PEG) hydrogel based polymers are among the most widely used synthetic materials for biomedical applications. Because of their biocompatibility, and ease of fabrication, hydrogels are highly suitable for use as constructs to engineer tissues as well as for cell transplantation. A critical parameter of importance for PEG hydrogels is their mechanical properties which are highly dependent on the environmental conditions. Properties of PEG-based hydrogels can be engineered to resemble scaffolds composed of extracellular matrix molecules, which provide structural support, adhesive sites and mechanical as well as biomechanical signals to most cells. The mechanical properties of these synthetic scaffolds can affect the migration, proliferation and differentiation of the cells. Accordingly, it is important to investigate the mechanical properties of these hydrogels and observe their effect on cell behavior as PEG-based scaffolds for example. In this research, the objective is to measure the mechanical properties such as the elastic modulus (Ec) and the stiffness (S) of polyethylene glycol diacrylate (PEGDA) hydrogel matrices at the nanoscale. The effect of varying parameters in the fabrication of PEGDA hydrogels including monomer molecular weight, initiator concentration and rates of hydration were investigated via nanoindentation using an atomic force microscope (AFM). Two different silicon nitride based cantilevers were used to study the effect of varying loading rates on the mechanical properties of these materials. Indentation parameters such as loads applied and indent depths were varied for each hydrogel sample. Different models were used to fit the experimental data to obtain the parameters of interest for the material (Ec and S). In particular, the data was best described using the model of Oliver-Pharr to analyze and fit the nanoindentation curves. Scanning electron microscope was used to image and confirm the geometry of the tip before and after the indentation experiments. Under high load and displacement modes, the indentation analysis was relatively easy and the elastic modulus and stiffness values were obtained for all dry PEGDA hydrogel sample. The variation of the initiator concentration has been analyzed as well. The mechanical properties of the hydrogel increase as the amount of the initiator increase in the precursor. The degree of hydration dramatically affects the mechanical behavior of the PEGDA. The presence of water within the hydrogel network weakens the internal as well the external mechanical properties, leading to smaller values of elastic modulus and stiffness compared with the dry condition. The mechanical properties of the indenter (cantilever tips) have significant impact on the results. It is important to study carefully the indenter properties before and after the indentation experiments. Since little work has been done on investigating the mechanical properties of PEGDA hydrogels at the nanoscale via AFM, the analysis of the mechanical behavior of this type of hydrogel using this strategy is of great importance

Nanomechanics of Electrospun Nanofibres for Tissue Engineering of the Tympanic Membrane

The Tympanic Membrane (TM), also known as the eardrum, includes layers of organized collagen nanofibres which play an essential role in sound transmission. Perforations that are caused by infection or accident must be repaired in order to restore hearing. Tympanoplasty is performed using grafts that are prepared from bladder, cartilage, temporal fascia and cadaveric skin. However, since mechanical properties of these grafts do not match those of the original TM, normal hearing is not fully restored. The goal of this study is to develop nanofibrous scaffolds for tissue engineering of the TM in order to circumvent the complications addressed with the conventional grafts. Mechanical properties of scaffolds greatly influence cellular behaviour, since cells can sense and respond to the stiffness of their substrate. In this study we investigated the Young’s modulus of single poly(caprolactone) (PCL) nanofibres as well as the moduli of as-spun and genipin-cross-linked collagen type I nanofibres using multi-point bending test with atomic force microscope (AFM). The effect of shear and tension on bending behaviour of fibres was investigated using four different analytical models. The Young’s modulus of electrospun PCL fibres (100 d 400 nm) was obtained with a mean value of 0.48 0.03 GPa. For as-spun and genipin-cross-linked collagen nanofibres a range of 1.66 – 13.9 GPa and 8.22 – 40.1 GPa were found for their Young’s moduli, respectively. The results indicate that there is a great potential for electrospun PCL and collagen nanofibres to be successfully applied in tissue engineering scaffolds because of their promising mechanical properties and biocompatibility

D,L-Cyclic Peptides as Structural Materials

The bioengineer has a choice of building with proteins, peptides, polymers, nucleic acids, lipids, metals and minerals, each class containing tremendous diversity within its category. While the platforms are diverse, they can be unified by a common goal: to engineer nano- and micro-scale order to improve functionality. In doing so, self-assembling systems aim to bring the lessons learned from the order in natural systems83 into the therapeutics, materials, and electronics that society uses every day. The rigid geometry and tunable chemistry of D,L-cyclic peptides make them an intriguing building-block for the rational design of nano- and microscale hierarchically structured materials. Herein, we utilize a combination of electron microscopy, nanomechanical characterization including depth sensing-based bending experiments, and molecular modeling methods to obtain the structural and mechanical characteristics of cyclo-[(Gln-D-Leu)4] (QL4) assemblies. QL4 monomers assemble to form large, rod-like structures with diameters up to 2 μm and lengths of 10s to 100s of μm. Image analysis suggests that large assemblies are hierarchically organized from individual tubes that undergo bundling to form larger structures. With an elastic modulus of 11.3 ± 3.3 GPa, hardness of 387 ± 136 MPa and strength (bending) of 98 ± 19 MPa the peptide crystals are among the most robust known proteinaceous micro- and nano-fibers. The measured bending modulus of micron-scale fibers (10.5 ± 0.9 GPa) is in the same range as the Young’s modulus measured by nanoindentation indicating that the robust nanoscale network from which the assembly derives its properties is preserved at larger length-scales. Materials selection charts are used to demonstrate the particularly robust properties of QL4 including its specific flexural modulus in which it outperforms a number of biological proteinaceous and non-proteinaceous materials including collagen and enamel. We then demonstrate a composite approach to mechanical reinforcement of polymeric systems by incorporating synthetic D,L-cyclic peptide nanotube bundles as a structural filler in electrospun poly D-, L-lactic acid fibers. With 8 wt% peptide loading, the composite fibers are >5-fold stiffer than fibers composed of the polymer alone, according to AFM-based indentation experiments. The facile synthesis, high modulus, and low density, and reinforcing capabilities of QL4 fibers indicate that they may find utility as a filler material in a variety of high efficiency, biocompatible composite materials. This study represents the first experimental mechanical characterization of D,L-cyclic peptide assemblies or composites.Engineering and Applied Sciences - Engineering Science