Nanolayers for early diagnostics of proteins involved in degenerative amyloidosis

Abstract

Abstract. FK-506 binding protein (FKBP12) is a protein of the family of immunophilins, involved in many neurodegenerative diseases such as Alzheimer's syndrome where FKBP12 is known to be over-expressed in early stages of the disease. We designed and built Langmuir-Blodgett nanostructures incorporating ligands with high affinity for FKBP12: Tacrolimus (FK506) and Rifaximin as candidate nanosensors to detect low FKBP12 concentration in the initial phase of the amyloidosis. The binding process of the different ligands has been studied by means of photophysical measurements investigating the fluorescence quenching of the tryptophan residue in the binding pocket of FKBP12 by addition of the ligand in solution. Immobilization of the ligands was achieved adopting biomimetic strategy: phospholipid Langmuir-Blodgett films are proposed as nanoscaffolds for ligand inclusion. Several phospholipid nanoarchitectures differing in lipid composition, fluidity, number of layers and method of production (incubation versus co-spreading) were screened. The results have shown that both FK506 and Rifaximin ligands penetrate the lipid matrix either as monomers or as aggregates depending on their initial concentration. More importantly, the experiments demonstrated that the ligands in the LB scaffolds efficiently quench FKBP12 fluorescence in solution as a consequence of ligand binding to the protein.