Design of a ligand-dependent fluorescent biosensor, based on an engineered lipocalin (Anticalin), for the sensitive detection of the Alzheimer β-amyloid peptide.

Based on the Anticalin H1GA which tightly binds Aβ40 and Aβ42 peptides - both established biomarkers of Alzheimer's disease - we describe the design of a protein-dye conjugate as analytical reagent that shows strongly elevated fluorescence upon Aβ binding. An unpaired Cys residue was introduced at seven positions within the four loop segments that shape the ligand pocket of the engineered lipocalin. Five of these mutants were purified in the monomeric state and allowed the site-specific conjugation with IANBD amide as a solvatochromic fluorophore. Three conjugates showed ligand-dependent fluorescence and one of these, derived from H1GA(D45C), exhibited sixfold higher emission at 546 nm upon complex formation with the peptide while revealing a low KD value of 1.2 ± 0.8 nM, even in the presence of 5 % (w/v) albumin. This NBD-conjugated Anticalin offers a novel biosensor with potential for the detection of Aβ peptides in biochemical assays or human body fluid samples.