Reducing uncertainties in energy dissipation measurements in atomic force spectroscopy of molecular networks and cell-adhesion studies

Atomic force microscope (AFM) based single molecule force spectroscopy (SMFS) is a valuable tool in biophysics to investigate the ligand-receptor interactions, cell adhesion and cell mechanics. However, the force spectroscopy data analysis needs to be done carefully to extract the required quantitative parameters correctly. Especially the large number of molecules, commonly involved in complex networks formation; leads to very complicated force spectroscopy curves. One therefore, generally characterizes the total dissipated energy over a whole pulling cycle, as it is difficult to decompose the complex force curves into individual single molecule events. However, calculating the energy dissipation directly from the transformed force spectroscopy curves can lead to a significant overestimation of the dissipated energy during a pulling experiment. The over-estimation of dissipated energy arises from the finite stiffness of the cantilever used for AFM based SMFS. Although this error can be significant, it is generally not compensated for. This can lead to significant misinterpretation of the energy dissipation (up to the order of 30%). In this paper, we show how in complex SMFS the excess dissipated energy caused by the stiffness of the cantilever can be identified and corrected using a high throughput algorithm. This algorithm is then applied to experimental results from molecular networks and cell-adhesion measurements to quantify the improvement in the estimation of the total energy dissipation.

Cryo-AFM development for high resolution DNA imaging and spectroscopy

Susana del Carmen Tobenas Borron

The thesis presents the development of a custom made atomic force microscope (AFM) conceived to image biological molecules at low temperatures in ultrahigh vacuum conditions, and its application to DNA single molecule studies. Starting from a first prototype working in contact mode, our aim was to implement non-contact AFM in order to reduce possible sample damaging due to strong tip interactions. The first chapter of the thesis discusses the technical solutions developed to improve the previous system. A complete setup for in situ exchange of samples and tips was designed and implemented. This permits a continuous operation of the microscope under the experimental conditions of UHV. Thermal instabilities and temperature differences between the tip and the sample were solved with the design of a movable shield to avoid heating by thermal radiation. The introduction of a new high voltage signal generator with variable frequency and the development of a user-friendly LabVIEW interface to communicate with this device notably improved the piezomotors driving. These piezomotors perform the micro-positioning of all the microscope parts and are driven through this interface. Finally, the previous tip-holder design was modified to incorporate a piezo actuator behind the cantilever, which provides the necessary excitation for the implementation of dynamic AFM modes. The installation of a Phase Lock Loop (PLL) circuit to lock the cantilever frequency and the replacement of old feedback electronics for a digital SPM Controller, with advanced options of frequency-shift feedback, was another requirement for the correct implementation of non-contact AFM. The second chapter deals with DNA sample preparation for high resolution imaging by AFM. Based on our studies, the advantages and drawbacks of the most commonly used techniques for DNA adsorption are discussed in detail. Our measurements in contact mode demonstrate the convenience of using hydrophobic substrates for high resolution DNA imaging. At hydrophilic surfaces, like mica, the presence of a thin water layer could be responsible of strong adhesion forces between the cantilever and the sample. In order to overcome this limitation we tested HOPG as hydrophobic substrate. HOPG, chemically modified with amphiphilic compounds, allowed us to deposit DNA without molecular damage or stress. The HOPG surface treatment was significantly improved. New protocols were developed to control the formation of dodecylamine disordered monolayers or lamella structures on the substrate. The lamella formation induces a subsequent self assembly of DNA single molecules. In the last chapter we discuss our results on DNA imaging obtained with the different operation modes implemented in our microscope: contact mode, tapping mode and frequency modulation (non-contact) AFM. We conclude that the last one provides higher signal stability and the necessary sensitivity to observe DNA details at a single-molecule level. We also present a study of frequency shift versus distance dependence (force spectroscopy experiments) performed in DNA samples. We discuss for the first time a plausible explanation to the origin of the atomic force contrast between the substrate and the DNA molecules. Force spectroscopy measurements performed along DNA molecules reveal two distinguishable types of interaction. This result suggests that the chemical composition of the minor-major groove and/or of the sugar-phosphate backbone along the molecule can be detected, opening numerous possibilities for further studies and applications of this technique.

EPFL2008

Ultra-light photovoltaic composite sandwich structures

Julien Rion

The ultra-light photovoltaic sandwich structure is a new multifunctional structure concept enabling weight and thus energy to be saved in high-tech solutions such as solar cars, solar planes or satellites. The novelty of this approach is to use solar cells as a load carrying element in the structure. The aim of this work was to investigate the failure mechanisms of such ultra-light sandwich structure and their correlation with microstructure, processing pressure, and strength in order to obtain optimal design and processing. To this end, composite sandwich structures were extensively studied with weights in the range of 650 – 850 g/m2, and comprising one 140 µm thick skin made of 0/90° carbon fiber-reinforced plastic (CFRP), one skin made of 130 µm thick mono-crystalline silicon solar cells, and a 29 kg/m3 honeycomb core. As a first step, core-to-skin bonding in a symmetric (CFRP / core / CFRP) sandwich, for which a design criterion was lacking, was especially studied. An adhesive deposition technique was developed enabling the adhesive weight used for core-to-skin bonding to be tailored. Based on adhesive contact angles, the formation of the adhesive fillets between honeycomb cell walls and skin was modeled. Core / skin debonding energy was measured and compared to core tearing energy measured with a new video-based method, and the failure mechanisms during skin peeling were investigated. It was thus ascertained that, to provide the highest debonding energy-to-weight ratio, the optimal adhesive weight was 35-40 g/m2. Furthermore, in contrast with classic sandwich structures with thicker skins, it was observed that the bending strength of the ultra-light sandwich panels increased with adhesive weight. This was due to the formation of adhesive fillets, which significantly increased the bending stiffness of the thin CFRP skin, and thus increased the compressive load causing local instability of the skin. Models taking into account the increased skin stiffness showed that the best adhesive quantity required to increase the strength-to-weight ratio was ∼40 g/m2. In a second step, the influence of processing pressure on the morphology and strength of symmetric (CFRP / core / CFRP) ultralight sandwich structures was investigated by using one-shot vacuum bag processing. This showed that higher processing pressures caused the formation of larger adhesive fillets and an increased waviness of the CFRP skin on vacuum bag side. These two effects had conflicting impacts on the strength of the structure. Waviness of the skin favored local instabilities, whereas adhesive menisci stabilized the skin. Modeling of the local instability of the skin by taking into account the waviness of the skin and the size of the menisci as a function of processing pressure enabled an optimal processing pressure of 0.7 bar to be identified, giving the highest strength-to-weight ratio. The third step of the study was devoted to the mechanical analysis of the mono-crystalline silicon solar cells. The brittle behavior of the cells was confirmed, and the Weibull failure probability curve was established with the mean tensile strength at 221 MPa. It was demonstrated by experimental testing and finite element modeling (FEM) that the low strength of the cells compared to the intrinsic strength of silicon (∼6.9 GPa) was not due to surface texturation of the cells used for increased efficiency, but to more severe surface or edge defects. FEM also showed that no significant reinforcing effect of the cells could be obtained with polymer encapsulation. In addition, thermo-mechanical stresses due to a mismatch of the coefficient of thermal expansion (CTE) between the Si cells and the polymer encapsulation were found to be negligible. In order to protect the cells against the environment, encapsulation of the cells was successfully carried out, using highly transparent fluoropolymer films treated with SiO2 plasma sputtering for improved adhesion, together with silicone adhesive. Finally, the integration of solar cells as a photovoltaic skin of an ultra-light sandwich structure was achieved using thin stress transfer ribbons to ensure load transfer between adjacent cells. It was observed that the cells were not damaged by sandwich panel processing, even in curved panels, thus showing that the processing windows of the different constituents were compatible. The asymmetric (Si / core / CFRP) photovoltaic sandwich structure with a weight equal to 800 g/m2 and a specific power density equal to ∼250 W/kg (i.e. 20 times more than standard commercial photovoltaic panels) demonstrated an equilibrated mechanical behavior, i.e. the CFRP skin, reinforcing ribbons, and solar cells had similar failure loads.

EPFL2008

Covalent attachment of proteins to calcite surface for the single molecule experiments

Two protocols of covalent attachment of proteins onto the calcite surface, viz. one using the metallochelat and second using the aminohexil, are elaborated. Single molecule force spectroscopy method has been used to test their efficiency and practical applicability. Experiments were performed measuring the specific interaction force between bovine serum albumin (BSA) fixed onto the freshly cleaved calcite single crystal surface (procedure under the study here) and its polyclonal antibody (Ab-BSA) immobilized onto an AFM tip using standard and well studied procedure. We found the conditions, when up to 3-3.5% of tip-sample approaches lead to the formation of a single specific bond. (C) 2007 Elsevier B.V. All rights reserved.

2008

Ultra-light photovoltaic composite sandwich structures

Julien Rion

EPFL2008

Cryo-AFM development for high resolution DNA imaging and spectroscopy

Susana del Carmen Tobenas Borron

EPFL2008