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. ii Résumé Les processus moléculaires basés sur les interactions spécifiques récepteur – ligand (réponse immune vers l'antigène étranger, communication entre les cellules nerveuses, etc.) sont essentielles pour bonne et stable performance d'organismes vivants. Le but de cette thèse est l'étude de ces processus moléculaires avec l'aide de la Microscopie de Force Atomique (AFM) opérée dans le mode de Spectroscopie de Force (FS). L'information comme la force et longueur de liaison ligand-récepteur peut être obtenue par des études FS standard où il n'y a pas d'excitation externe appliquée sur les leviers. Dans un premier temps, …