Increasingly used in industrial coatings, polyelectrolytes multilayers (PEMs) are self-assembled systems made of the alternate deposition of oppositely charged polymers on substrates, usually built by the traditional layer-by-layer method. Their properties strongly depend on environmental physico-chemical parameters. Due to the variety of conditions used in the literature on the one hand and the diversity of polyelectrolytes systems on the other hand, it remains difficult to bring out general principles, leading now to a lack of a real understanding of the PEM buildup, from the macro- to the nanoscale. Here, combining acoustic and electrochemical methods with atomic force microscopy, in a systematic approach, we uncover the critical role of the deposition protocol in the growth regime of PEMs made of cationic poly (allylamine hydrochloride) and anionic poly(4-styrene sulfonate, sodium). Traditional dipping leads to thick, heterogeneous and relatively isolating PEMs whereas a spin-coating assisted method leads to thinner, homogeneous and more permeable PEMs. We also highlight that the pH and the ionic strength influence not only the electrostatic interactions and polyelectrolyte conformation in solution but also their organization after their adsorption on the substrate. Finally, our easily and rapidly adaptable protocol paves the way for promising potential bio-applications, since PEMs are applied to the bacterial immobilization on substrates or as a coating for nanostructured biosensor transducer.< Leer menos