Chiral 8-aminoBODIPY-based fluorescent probes with site selectivity for the quantitative detection of HSA in biological samples

Abstract

Human serum albumin (HSA) is one of the vital proteins in blood serum, and its optimum level is a reflection of the physiological well-being of an individual. Any abnormalities in serum HSA levels could often be a sign of disguised physiological disorders. The importance of fast and accurate determination of serum HSA levels has led to the development of various quantification methods. Among these, fluorescence-based methods employ molecular probes capable of producing selective responses on interaction with HSA. Herein, we report chiral 8-aminoBODIPY-based probes having blue emission for the quantitative detection of HSA in buffer and human blood serum. A pair of 8-aminoBODIPY enantiomers, namely R-PEB and S-PEB, were synthesized. They exhibited a fast ‘turn-on’ fluorescence response towards HSA, allowing its detection and quantification. In PBS buffer, R-PEB and S-PEB showed very good sensitivity with a limit of detection (LoD) of 25 nM (K_D = 9.84 ± 0.14 μM) and 39 nM (K_D = 18.67 ± 0.21 μM), respectively. The linear relationship observed between the fluorescence intensity of R-PEB/S-PEB and the HSA concentration in serum samples allowed us to generate a reference curve for HSA estimation for practical applications. Examination of unknown serum samples showed a good correlation with the results obtained by the benchmark BCG method. Interestingly, the difference in these probes’ dissociation constants and LoD indicated their differential binding to HSA. Considering the availability of multiple ligand binding sites in HSA, their binding preferences were investigated in detail by displacement assays using site-specific drugs. These studies showed the preferential affinity of R-PEB towards site II, which was further substantiated using molecular docking studies. However, these displacement assays could not identify the preferred binding site of S-PEB. Blind docking studies indicated that S-PEB occupied a site closer to FA5. Selective binding of R-PEB to site II and its characteristic photophysical response can be utilized to quickly screen potential site II binding drugs.