The quantification of interaction stoichiometry and binding constant between bacteria (or other microorganism) and (macro)molecules remains a challenging issue for which only a few adapted methods are available. In this paper, a new methodology was developed for the determination of the interaction stoichiometry and binding constant between bacteria and (macro)molecules. The originality of this work is to take advantage of the bacterial aggregation phenomenon to directly quantify the free ligand concentration in equilibrated bacteria-ligand mixtures using frontal analysis continuous capillary electrophoresis. The described methodology does not require any sample preparation such as filtration step or centrifugation. It was applied to the study of interactions between Erwinia carotovora and different generations of dendrigraft poly-L-lysines leading to quantitative information (i.e., stoichiometry and binding site constant). High stoichiometries in the order of 10(6)-10(7) were determined between nanometric dendrimer-like ligands and the rod-shaped micrometric bacteria. The effect of the dendrimer generation on the binding constant and the stoichiometry is discussed. Stoichiometries were compared with those obtained by replacing the bacteria by polystyrene microbeads to demonstrate the internalization of the ligands inside the bacteria and the increase of the specific surface via the formation of vesicles.
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