Two-Photon Macromolecular Probe Based on a Quadrupolar Anthracenyl Scaffold for Sensitive Recognition of Serum Proteins under Simulated Physiological Conditions

[Image: see text] The binding interaction of a biocompatible water-soluble polycationic two-photon fluorophore (Ant-PIm) toward human serum albumin (HSA) was thoroughly investigated under simulated physiological conditions using a combination of steady-state, time-resolved, and two-photon excited fluorescence techniques. The emission properties of both Ant-PIm and the fluorescent amino acid residues in HSA undergo remarkable changes upon complexation allowing the thermodynamic profile associated with Ant-PIm–HSA complexation to be accurately established. The marked increase in Ant-PIm fluorescence intensity and quantum yield in the proteinous environment seems to be the outcome of the attenuation of radiationless decay pathways resulting from motional restriction imposed on the fluorophore. Fluorescence resonance energy transfer and site-marker competitive experiments provide conclusive evidence that the binding of Ant-PIm preferentially occurs within the subdomain IIA. The pronounced hypsochromic effect and increased fluorescence enhancement upon association with HSA, compared to that of bovine serum albumin (BSA) and other biological interferents, makes the polymeric Ant-PIm probe a valuable sensing agent in rather complex biological environments, allowing facile discrimination between the closely related HSA and BSA. Furthermore, the strong two-photon absorption (TPA) with a maximum located at 820 nm along with a TPA cross section σ(2) > 800 GM, and the marked changes in the position and intensity of the band upon complexation definitely make Ant-PIm a promising probe for two-photon excited fluorescence-based discrimination of HSA from BSA.