Adsorption of biopolymers and their layer-by-layer assemblies on hydrophilic surfaces

Abstract: It is widely known that surfaces play an important role in numerous biological processes and technological applications. Thus, being able to modify surface properties provides an opportunity to control many phenomena occurring at interfaces. One way of controlling surface properties is to adsorb a polymer film onto the surface, for example through layer-by-layer (LbL) deposition of polyelectrolytes. This simple but versatile technique enables various polymers, proteins, colloidal particles etc. to be incorporated into the film, resulting in a multifunctional coating. Due to recent legislations and a consumer demand for more environmentally friendly products, we have chosen to use natural polymers (biopolymers) from renewable resources. The focus of this thesis has been on the adsorption of biopolymers and their layer-by-layer formation at solid-liquid interfaces; these processes have been studied by a wide range of techniques. The main method was the quartz crystal microbalance with dissipation monitoring (QCM-D), which measures the adsorbed mass, including trapped solvent and the viscoelastic properties of an adsorbed film. This technique was often complemented with an optical method, such as ellipsometry or dual polarization interferometry (DPI), which provided information about the “dry” polymer or protein adsorbed mass. From this combination, the solvent content and density of the layers was evaluated. In addition, the surface force apparatus (SFA), X-ray photoelectron spectroscopy (XPS), total internal reflection fluorescence (TIRF), and fluorescence resonance energy transfer (FRET) were utilized, providing further information about the film structure, chemical composition, and polymer inter-layer diffusion. Adsorption studies of the glycoprotein mucin, which has a key role in the mucousal function, showed that despite the net negative charge of mucin, it adsorbed on negatively charged substrates. The adsorbed layer was highly hydrated and the segment density on the substrate was low. We showed the importance of characterizing the mucin used, since differences in purity, such as the presence of albumin, gave rise to different adsorption behaviours in terms of both adsorbed amount and structure. The adsorbed mucin layer was to a large extent desorbed upon exposure to the anionic surfactant sodium dodecyl sulfate (SDS). In order to prevent desorption, we demonstrated that a protective layer of the cationic polysaccharide chitosan could be adsorbed onto the mucin layer and that the mucin-chitosan complexes resisted the desorption normally induced by association with SDS. Moreover, the association between chitosan and SDS was examined at the solid-liquid interface, in the bulk, and at the air-water interface. In all these environments chitosan-SDS complexes were formed and a net charge reversal of the complexes from positive to negative was observed when the concentration of SDS was increased. Furthermore, the LbL deposition method could be used to form a multilayer-like film by alternate adsorption of mucin and chitosan on silica substrates. The LbL technique was also applied to two proteins, lysozyme and β-casein with the aim of building a multilayer film consisting entirely of proteins. These proteins formed complexes at the solid-liquid interface, resulting in a proteinaceous layer, but the build-up was highly irregular with an increase in adsorbed amount per protein deposition cycle that was far less than a monolayer.Continuing with chitosan, known to have antibacterial properties we assembled multilayers with an anti-adhesive biopolymer, heparin, to evaluate the potential of this system as a coating for medical implants. Multilayers were assembled under various solution deposition conditions and the film structure and dynamics were studied in detail. The chitosan-heparin film was highly hydrated, in the range 60-80 wt-% depending on the deposition conditions. The adsorbed amount and thickness of the film increased exponential-like with the number of deposition steps, which was explained by inter-diffusion of chitosan molecules in the film during the build-up. In a novel approach, we used the distant dependent FRET technique to prove the inter-layer diffusion of fluorescent-labelled chitosan molecules within the film. The diffusion coefficient was insignificantly dependent on the deposition pH and ionic strength, and hence on the film structure. With the use of a pH sensitive dye buried under seven chitosan-heparin bilayers, we showed that the dye remained highly sensitive to the charge of the outermost layer. From complementary QCM-D data, we suggested that an increase in the energy dissipation does not necessarily indicate that the layer structure becomes less rigid.