Live bacterial cells are used in many biotechnological and biomedical applications that often require modifications of the cellular surface. A possible method for cell surface modification is nanocoating via layer-by-layer method, which is based on electrostatically driven deposition of polyelectrolytes onto a charged cell surface. We observed the formation of the polycation layer on a negatively charged bacterial cell surface. This is the first master thesis studying nanocoating of cells with stimulated emission depletion microscopy (STED).
The aim of this work was to optimize a protocol for fluorescent labelling of polycations with a fluorescent dye and determine the appropriate dye, manifesting an improvement in resolution on the used STED microscope when compared to confocal microscope. A systematic study of nanocoating of two bacterial species (E. coli and P. stutzeri) was done by measuring zeta potential (ZP) and observing the nanocoated cells with STED. We also followed the nanocoating process with isothermal titration calorimetry (ITC).
STAR RED showed an improved resolution in STED when compared to confocal microscopy, whereas ATTO 594 and rhodamine-B-isothiocyanate did not. We labelled polycations (high and low molecular weight polyethyleneimines - PEI and polyallylamine hidrochlorides - PAH) with STAR RED and used them to coat E. coli and P. stutzeri. STED microscopy showed the formation of a polyelectrolyte layer on the cell surface. Polycations permeated into P. stutzeri and not into E. coli. We did not observe a difference in cell wall penetration between high and low molecular weight polymers. ZP changed from negative before to positive after nanocoating. ITC termograms, showing the dependance of heat rate on the amount of added polymer, reach a plateau in the case of E. coli, suggesting achievement of cell surface saturation with polycation. In case of P. stutzeri the curve is not the expected sigmoidal shape as polycations permeate into the cell.
We conclude the nanocoating of studied bacterial cells with PEI and PAH was successful, as surface charge changed from negative to positive. Permeation of polycations through the cell wall depends less on molecular weight and more on species, which was proven by observing the localization of fluorescently labelled polycations after nanocoating with STED. ITC confirmed the saturation of the cellular surface with the polycation.