Noncovalent complexes of anionic carbocyanine dyes with human serum albumin as a basis for turn-off fluorescence sensors for bilirubin. Effect of Cu2+ ions

Abstract

The development of highly sensitive and selective turn-off fluorescence sensors for bilirubin (BR) is of significant clinical importance. This work explores the basis for such sensors using the noncovalent complexes of meso-substituted anionic carbocyanine dyes with human serum albumin (HSA). The binding of these dyes to HSA is characterized by high association constants (of the order of 10⁵–10⁶ M⁻¹) and induces a pronounced fluorescence increase due to the stabilization of monomeric trans-isomers. Subsequent introduction of BR, which itself binds strongly to HSA, results in efficient fluorescence quenching (super-quenching effect). Comprehensive spectral studies and molecular docking simulations indicate that the primary quenching mechanism is the competitive displacement of dye molecules from the complex with HSA into the weakly fluorescent free state. The quenching effect is somewhat enhanced in the presence of Cu²⁺ ions. The effective Stern–Volmer quenching constants were determined, and the analytical potential of these systems was evaluated, revealing low limits of detection (LOD) and quantification (LOQ) of BR. The dye–HSA and dye–HSA–Cu²⁺ complexes presented herein thus create promising and efficient platforms for the rational design of turn-off sensors for BR.