Benzothiazole based cyanine dyes with bridged groups in pentamethine chain were studied as potential far-red fluorescent probes for protein detection. Spectral-luminescent properties were characterized for unbound dyes and in the presence of serum albumins (bovine (BSA), human (HSA), equine (ESA)), and globular proteins (β-lactoglobulin, ovalbumin). We have observed that the addition of albumins leads to a significant increase in dyes fluorescence intensity. However, the fluorescent response of dyes in the presence of other globular proteins was noticeably lower. The value of fluorescence quantum yield for dye bearing sulfonate group complexed with HSA amounted to 42% compared with 0.2 % for the free dye. The detection limit of HSA by this dye was about > 0.003 mg/ml that indicates the high sensitivity of dye to low HSA concentrations. Modeling of structure of the dyes complexes with albumin molecules was performed by molecular docking. According to these data, dyes could bind to up to five sites on the HSA molecule; the most preferable are the hemin-binding site in subdomain IB and the dye-binding site in the pocket between subdomains IA, IIA, and IIIA. This work confirms that pentamethine cyanine dyes could be proposed as powerful far-red fluorescent probes applicable for highly sensitive detection of albumins.

Spectral measurements. Spectroscopic measurements were performed in standard quartz cuvette (10x10 mm). Fluorescence excitation and emission spectra were registered using the fluorescent spectrophotometer Cary Eclipse (Varian, Australia). Absorption spectra were registered using the spectrophotometer Genesys 20 (ThermoScientific). All the spectral-luminescent characteristics of dyes were studied at room temperature. Direct datasets from the devices for absorption and fluorescence measurments is loaded. 

Quantum yield determination. The quantum yield value of the dyes 2 and 3 free and in the presence of serum albumins (HSA and BSA, respectively) was determined using Nile Blue (NB) solution in methanol as the reference (quantum yield value φ_NB = 0.27) [17]. Namely, solutions of dye 2 and dye 3 free, dye 2 in the presence of BSA, dye 3 in the presence of HSA (all in buffer) and Nile Blue in methanol were taken in such concentrations that their optical density values were equal at 645 nm. Fluorescence of all these solutions was excited at this wavelength (645 nm), and the area below each spectrum (S_dye for dyes 2 and 3 and S_NB for Nile Blue) was calculated. Further, the fluorescence quantum yield φ_dye of dye 2 and dye 3 free, dye 2 in the presence of BSA, dye 3 in the presence of HSA was calculated as: φ_dye = φ_NB×(S_dye/S_NB)×(n_H2O/n_meth)², where n_H2O and n_meth are refractive indexes of water and methanol respectively. Quantum yiled for Dye2 and Dye3 was calculated using Origin program, the corresponding Origin project contating formulas for φ calculation is loaded. Direct datasets from the devices for absorption and fluorescence measurments is also provided. 

Molecular docking. Molecular docking of fluorescent dyes to the entire protein surface of human serum albumin (crystal structure with PDB accession code 1AO6 [18]) and equine serum albumin (PDB ID: 4F5U [19]) was performed using CB-Dock web-server (http://cao.labshare.cn/cb-dock/) [20]. Water molecules were removed from the PDB file of receptor. Blind molecular docking was performed into five binding sites. The complexes were visually inspected using Discovery Studio Visualizer 4.0 [21]. Dataset of results is loaded as HSA_Molecular docking_results.rar file.