Can Dissipative Properties of Single Molecules Be Extracted from a Force Spectroscopy Experiment?

We performed dynamic force spectroscopy of single dextran and titin 127 molecules using small-amplitude and low-frequency (40-240 Hz) dithering of an atomic force microscope tip excited by a sine wave voltage fed onto the tip-carrying piezo. We show that for such low-frequency dithering experiments, recorded phase information can be unambiguously interpreted within the framework of a transparent theoretical model that starts from a well-known partial differential equation to describe the dithering of an atomic force microscope cantilever and a single molecule attached to its end system, uses an appropriate set of initial and boundary conditions, and does not exploit any implicit suggestions. We conclude that the observed phase (dissipation) signal is due completely to the dissipation related to the dithering of the cantilever itself (i.e., to the change of boundary conditions in the course of stretching). For both cases, only the upper bound of the dissipation of a single molecule has been established as not exceeding 3.10(-7) kg/s. We compare our results with previously reported measurements of the viscoelastic properties of single molecules, and we emphasize that extreme caution must be taken in distinguishing between the dissipation related to the stretched molecule and the dissipation that originates from the viscous damping of the dithered cantilever. We also present the results of an amplitude channel data analysis, which reveal that the typical values of the spring constant of a 127 molecule at the moment of module unfolding are equal to 4 +/- 1.5 mN/m, and the typical values of the spring constant of dextran at the moment of chair-boat transition are equal to 30 - 50 mN/m.

Cartographie et spectroscopie des propriétés mécaniques à l'échelle du nanomètre par spectrométrie acoustique locale à fréquence variable

This work aims at completing the development of local mechanical spectroscopy as initiated by F. Oulevey during his thesis. More precisely, the measurement of the amplitude and phase lag of the strain as a function of the applied stress frequency bas to be settled. Parallel to the development of the technical aspects making such an acquisition possible, the model used to analyze the measured quantities is modified in order to describe the studied system more realistically. The local mechanical spectrometer is based on an Atomic Force Microscope (AFM) using an optical method to detect the deflection of the lever. The stress is applied locally on the sample by a transducer fixed at the base of the cantilever. The bandwidth of the microscope' s electronics does not allow one to detect the cantilever movement at frequencies higher than the MHz. Two different approaches are followed to overcome this limit. The first approach exploits the nonlinearity of the contact to down-convert, via mechanical mixing, the high-frequency signals into low-frequency ones. This work demonstrates the feasibility of this technique, as well as its limits, mainly due to the difficulty to analyze confidently the obtained results. The second approach takes advantage of the stroboscopic detection of the movement of the cantilever. The intensity of the laser used to detect the deflection of the cantilever is modulated at a frequency close to the applied stress frequency. The amplitude of the signal recorded at the difference frequency corresponds then to the high-frequency amplitude of the cantilever movement. The frequency range attainable with this method is only limited by the bandwidth of the transducer providing the excitation. The model connecting the measured quantities (strain amplitude and phase lag) to the desired mechanical properties (elasticity and damping) consists in a beam clamped at one end, with a tip attached at some point along its length. The tip itself is connected to the sample via a spring and a dashpot in parallel, representing the normal interaction, and a spring and a dashpot in parallel representing the interaction in the plane of the sample. Combined with the stroboscopic detection, this model enables one to quantitatively determine the elastic modulus of stiff samples. Many results obtained on a wide range of samples exhibiting very diverse mechanical properties are presented. They demonstrate the validity of the stroboscopic approach, which combines simplicity of use and efficiency.

EPFL2000

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

Quasistatic and dynamic force microscopy of single antigen-antibody complexes and fibrin-fibrinogen systems

The molecular processes based on the specific receptor-ligand interactions (e.g. immune response to a foreign antigen, communication between nerve cells, etc.) are essential for a good and stable performance of living organisms. The aim of the present work is to study such molecular processes with the Atomic Force Microscope (AFM) operated in Force Spectroscopy (FS) mode. Information such as the force and length of the ligand-receptor bond rupture could be obtained from the standard quasistatic FS studies, whereas no external dithering applied to the AFM tips. Firstly, the antigen-antibody interactions were examined with the Bovine Serum Albumin (BSA) protein and its poly- and two different monoclonal antibodies. It was shown that the peak unbinding force depends on the type of antibody and the antibody concentration. The single potential barrier is dominant for the interaction between BSA and monoclonal antibodies and was revealed from the loading rate-dependent measurements. Secondly, we are interested in the process of fibrin gel formation, in particular in the forces involved in this process. It was demonstrated that the fibrin-fibrinogen interaction is the specific one. The unbinding force rupture for single fibrin-fibrin(ogen) complex was determined to be of about 320 pN at a loading rate of 3.5 nN/s. The single potential barrier is also dominant for this interaction. Moreover, a new method of direct and continuous measurement of the spring constant of single molecules or molecular complexes has been elaborated in our laboratory. To this end, the standard FS technique with functionalized tips and samples is combined with a small dithering of the AFM tip. The change of the dithering amplitude as a function of the pulling force is measured in order to extract the spring constant of the complex. The potentialities of this technique were demonstrated for the experiments with single BSA – polyclonal antibody to BSA (Ab-BSA) and fibrinogen – fibrinogen complexes.

EPFL2004