Electrochemical sensing and imaging of biological samples

Implementation of analytical methods for biological samples (e.g. bacteria or mammalian cells) is of great importance for diagnosis and treatment of various diseases, as well as for environmental monitoring. However, analysing biological samples is a cumbersome task since it requires a high sensitivity and selectivity, a capability to sense dynamic and spatial concentration changes and time-consuming separation strategies. Although several approaches have been proposed to address all these points, there is still room for the exploration and development of new analytical platforms that contribute to the manipulation and understanding of biological systems. The aim of the present thesis is therefore to propose, characterize and implement new analytical tools that can be used for perturbing, sensing and imaging of biological samples such as adherent cancer cells. As a first approach, scanning electrochemical microscopy (SECM) was combined with cell fixations strategies in order to investigate differences between adherent melanoma cells corresponding to various cancer stages. For this purpose, alive, fixed and permeabilized cells were characterized electrochemically and additionally immunostained in order to visualize by SECM the different intracellular distribution of tyrosinase among three melanoma cancer stages. Constant height SECM is widely used for cells investigation, but the intricate cells topography can influence significantly the real probe-substrate distance and thus encumber the interpretation of the obtained experimental data. Applying soft stylus probes in a contact mode could be an alternative to alleviate such a limitation, although it can also introduce damages on the biological samples. Therefore, as the next step the applicability of the soft stylus concept towards the scanning of adherent living cells in a contact mode was investigated. As a result, modified ultra-soft probes were implemented for the successful and damage-free contact mode scanning of adherent melanoma cells. While the most of the SECM experiments with adherent cells have been devoted to the â readingâ of a biological response, the perturbation of the cell microenvironment through spatially localized electrochemical or chemical reactions is also of high importance. Therefore, the SECM soft stylus concept was extended as a tool for locally altering the microenvironment of few adherent living cells. A re-designed electrochemical push-pull probe was employed for controlling the extracellular space of a small number of adherent cells. Besides SECM, mass-spectrometry (MS) can be also used for cancer cells characterisation. Therefore, in this thesis an intact cell matrix-assisted laser desorption/ionization MS approach was combined with various cell fixation techniques in order to obtain a simple and fast protocol for acquiring the MS fingerprint of cancer cells. As a result, the developed protocol allowed the characterization and differentiation of various melanoma