Decoupling viscosity induced fluorescence from HSA-induced absorption modulation in a chromyl-naphthyl molecular...

Molecular rotors are valuable probes for studying local microenvironments because their optical behavior is closely linked to conformational dynamics. In this study, a chromyl-naphthyl (HCNP) donor-π-acceptor molecular rotor was developed for viscosity-responsive fluorescence and selective interaction with human serum albumin (HSA). HCNP showed weak fluorescence in low-viscosity media, whereas glycerol produced a 12-fold enhancement in emission intensity. The viscosity-dependent emission, supported by the Förster-Hoffmann relationship (R² = 0.97) and temperature-dependent fluorescence studies, confirms that emission enhancement arises from the restriction of intramolecular rotation. Unlike glycerol, HSA didn't produce a noticeable fluorescence "turn-on" response. In contrast, the addition of HSA produced a selective red shift in the UV-Vis spectrum of HCNP, shifting the absorption maximum from 395 to 445 nm. This spectral change, which appeared within 10 s, was not observed with other tested biomolecules/interfering analytes. Absorption titration confirmed the formation of the HCNP-HSA (1 : 1) complex with an association constant of 3.75 × 10⁵ M^-1. Molecular docking suggested preferential binding of HCNP at Sudlow's site I near the Trp214 residue with a docking score of -10.39 kcal mol^-1. Molecular dynamics simulation further supported the complex stability, which was mainly driven by non-covalent interactions. DFT calculations further revealed that the twisted/non-planar conformer has a smaller energy gap than the planar form, supporting the experimentally observed HSA-induced red shift. These findings establish HCNP as a dual photophysical probe in which viscosity is reported via fluorescence enhancement, while HSA recognition is expressed via absorption modulation caused by protein-induced conformational and electronic stabilization.