The mechanism of formation of supported lipid layers from phosphatidylcholine and phosphatidylserine vesicles in solution on polyelectrolyte multilayers was studied by a variety of experimental techniques. The interaction of zwitterionic and acidic lipid vesicles, as well as their mixtures, with polyelectrolyte supports was followed in real time by micro-gravimetry. The fabricated lipid-polyelectrolyte composite structures on top of multilayer coated colloidal particles were characterized by flow cytometry and imaging techniques. Lipid diffusion over the macroscopic scale was quantified by fluorescence recovery after photobleaching, and the diffusion was related to layer connectivity. The phospholipid-polyelectrolyte binding mechanism was investigated by infrared spectroscopy. A strong interaction of polyelectrolyte primary amino groups with phosphate and carboxyl groups of the phospholipids, leading to dehydration, was observed. Long-range electrostatic attraction was proven to be essential for vesicle spreading and rupture. Fusion of lipid patches into a homogeneous bilayer required lateral mobility of the lipids on the polyelectrolyte support. The binding of amino groups to the phosphate group of the zwitterionic lipids was too weak to induce vesicle spreading, but sufficient for strong adsorption. Only the mixture of phosphatidylcholine and phosphatidylserine resulted in the spontaneous formation of bilayers on polyelectrolyte multilayers. The adsorption of phospholipids onto multilayers displaying quarternary ammonium polymers produced a novel 3D lipid polyelectrolyte structure on colloidal particles.

Cholesterol-based lipophilic oligonucleotides incorporated into lipid membranes were studied using solid-state NMR, differential scanning calorimetry, and fluorescence methods. Lipophilic oligonucleotides can be used to build nanotechnological structures on membrane surfaces, taking advantage of the specific Watson-Crick base pairing. We used a cholesteryl-TEG anchor first described by Pfeiffer and Hook (J. Am. Chem. Soc. 2004, 126, 10224-10225). The cholesterol-based anchor molecules were found to incorporate well into lipid membranes without disturbing the bilayer structure and dynamics. In contrast to cholesterol, which is known to induce significant condensation of the membrane lipids, the cholesteryl-TEG anchor does not display this property. When the cholesteryl-TEG moiety was covalently bound to an oligonucleotide, the resulting lipophilic DNA molecules inserted spontaneously into lipid membranes without altering their structure. The duplex formed by two complementary cholesteryl-TEG oligonucleotides had increased thermodynamic stability compared to the same oligonucleotides without the anchor, both in solution and incorporated into lipid membranes. Since the cholesteryl-TEG anchor lacks the characteristic properties of cholesterol, oligonucleotides modified with this anchor are equally distributed between liquid-disordered and liquid-ordered domains in "raft" forming membranes. As an example of an application of these lipophilic oligonucleotides, cholesteryl-TEG-DNA was incorporated into supported lipid bilayers formed on polyelectrolyte-coated silica microparticles. The modified oligonucleotides were stably inserted into the lipid membrane and retained their recognition properties, therefore enabling further functionalization of the particles.

We assembled multilayer films of glucose oxidase (GOx) and horseradish peroxidase (HRP) coimmobilized together with polyelectrolyte layers on the surface of silica microparticles. The influence of different polyelectrolyte combinations on the immobilization and functionality of the enzymes was examined for several multilayer configurations. Precomplexation of the enzymes with a polyvinylpyridine-based polyamine allowed the stable adsorption of enzyme layers without affecting their catalytic activity. The efficiency of the sequential reaction between GOx and HRP on the surface of the colloids was quantitatively analyzed and rationalized in terms of the kinetic parameters of both enzymes and the reaction-diffusion kinetics of the system. In the optimized configuration, with GOx and HRP coimmobilized in the same layer, the overall rate of hydrogen peroxide conversion was around 2.5 times higher than for GOx and HRP in separate layers or for equivalent amounts of both enzymes free in solution.