Understanding selective sensing of human serum albumin using a D–π–A probe: a photophysical and computational approach†

The human serum albumin (HSA) level is a valuable indicator of an individual's health status. Therefore, its detection/estimation can be used to diagnose several diseases. In this work, we have developed a series of donor–π–acceptor probes, which were found to selectively detect HSA over BSA (bovine serum albumin). Among these probes, A4, which bears the trifluoroacetyl group, showed the highest selectivity for HSA, with limits of detection and quantification being 1.36 nM and 2.59 nM, respectively. CD spectroscopy of the HSA–A4 ensemble indicated an increase in the α-helicity of the protein, while the displacement assays revealed the localization of the probe in the hemin site of HSA. The probe works on the principle of excited state intramolecular charge transfer (ICT). Its selectivity was also validated computationally. Docking experiments confirmed the preference of the probe for the hemin binding IB site of HSA, as observed from the fluorescence displacement assay results, and a comparison of docking scores demonstrated the greater preference of A4 for HSA compared to BSA. Computational experiments also showed a change in preference for HSA amino acid residues exhibited by the excited state of probe A4 (Tyr161, Met123, Pro118, and Leu115) when compared to its ground state (Arg186 and His146). Hydrophobic interactions dominated the excited state protein–probe ensemble, whereas there was significant involvement of the water bridges along with the hydrophobic interactions in the ground state ensemble. Probe A4 was also assessed for its practical utility and found to successfully sense HSA in urine at extremely low concentrations. Moreover, the A4–HSA ensemble was employed for hemin sensing with a detection limit of 0.23 μM.