Faculté des sciences

Nanotools for combined AFM-SECM experiments in structural biology

Gullo, Rosario Maurizio ; Staufer, Urs (Dir.)

Thèse de doctorat : Université de Neuchâtel, 2006 ; 1915.

Many important biological activities like electrical impulses in nerve cells and cell metabolism are strongly related to the transport of molecules into and out of biological cells. Consequently, there is great interest in understanding these cellular transport mechanisms. The patch clamp technique gives a quantitative insight in cellular transport. This method generally uses a glass pipette to... Plus

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    Summary
    Many important biological activities like electrical impulses in nerve cells and cell metabolism are strongly related to the transport of molecules into and out of biological cells. Consequently, there is great interest in understanding these cellular transport mechanisms. The patch clamp technique gives a quantitative insight in cellular transport. This method generally uses a glass pipette to measure the electrochemical current across the cell membrane. Atomic force microscopy (AFM), on the other hand, has been applied to study the topography of membranes. To get a quantitative and qualitative comprehension of the cellular transport it would be advantageous to observe the topographic and local electrical information simultaneously. This could be achieved by a setup that combines the conventional patch clamp technique and AFM. However, the combination and implementation of these two techniques requires the contrivance of new tools. This thesis is devoted to the fabrication and characterization of such tools. In a first step the glass pipette used in patch clamp has been replaced by a planar sample support, which is more adapted to the requirements of AFM. The supports feature a nanometer scaled aperture in order to enable measurements on suspended membrane patches. Their electrical, mechanical and biological characteristics were investigated and designed to fit the requirements of the aimed application. In order to keep the biological cells functional, the experiments need to be performed in a liquid environment. Therefore a special conductive AFM probe allowing local electrical measurements in liquid environment has been designed and realized. The probe is electrically insulated except at the very apex of the tip, which has a radius of curvature smaller than 10nm. The imaging quality of the probe has been assessed and nanometer lateral resolution on biological membranes has been achieved. The electrochemical behavior of the tip has been investigated by cyclic voltammetry. Moreover the probes were employed to perform combined AFM and scanning electrochemical microscopy (SECM) experiments. It was possible to simultaneously acquire the topography and the electrochemical current images with a lateral resolution below 10nm. Measurements through the submicron scaled aperture in the planar sample support were used to investigate the distance dependency of the electrochemical current. First measurements on biological cells absorbed on the planar sample support showed an excellent correlation between topography and electrochemical current. In order to gain a deeper insight into the relation between the tip geometry and the resulting electrochemical current and imaging resolution, a finite element simulation model has been built. The simulations agreed with the measurements and theory. This results proved the accuracy of the finite element model.