A fluorescent "Turn-ON" probe with rapid and differential response to HSA and BSA: quantitative detection of HSA in urine.

The present study provides insight into the differential response of a benzimidazole-malononitrile fluorescent "Turn-ON" probe on interaction with two structurally similar proteins, BSA and HSA. Compound 6 shows more sensitivity towards the two SAs, which is completely lost in the case of compound 7, synthesized by substitution on 6. The aggregates of compound 6 show absorption maxima at 385 nm and weak emission maxima at 565 nm. Compound 6 forms a new emission band at 475 nm on gradual addition of BSA (200 μM) along with a slight increase in the emission band at 565 nm. However, on addition of HSA (50 μM), a new band at 475 nm is formed. In contrast to BSA, in the case of HSA, 50% quenching is observed in the emission band of compound 6 at 565 nm. The new band formed on the interaction of 6 with BSA shows four-fold more enhancement compared to HSA. Furthermore, the mechanism of interaction of 6 with serum albumin has been investigated through lifetime-fluorescence analysis, site-selective drug experiments, dynamic light scattering, FE-SEM, FT-IR, etc. Molecular docking studies and site marker drug displacement experiments reveal differential interactions of 6 towards the two structurally similar proteins. Aggregates of 6 with an average hydrodynamic size of 100-190 nm are disassembled on adding BSA and HSA, and the size of the serum albumin and 6 complex decreases to 10-20 nm, revealing the ligand's encapsulation in the serum albumin cavity. Practical applicability for the quantitative detection of HSA in human urine samples is also demonstrated. The high binding affinity, sensitivity, selectivity and differential response of probe 6 towards two serum albumins (HSA and BSA) and significant quantification of HSA in urine samples shows the potential ability of this probe in medical applications.