Clarifying the role of carboxyl functionality in squaraines towards aggregation and self-assembly through photophysical characterization and BSA interactions

Abstract

The demand for near-infrared (NIR) probes with robust and stable fluorescence signals has surged, driven by their applications in optical sensing and bioimaging. Squraine dyes as NIR fluorescent probes have gained significant traction. However, understanding their behavior in aqueous media is important for their successful application. Herein we report the synthesis and photophysical characterization of carboxy-functionalized unsymmetrical squaraine dyes capable of light absorption and emission in the near-infrared (NIR) wavelength region. Structural variations in alkyl chain length, and spacer chain length between the primary chromophore and –COOH group were systematically introduced to investigate their impact on self-assembly in aqueous media leading to dye aggregation and their differential interactions with Bovine Serum Albumin (BSA) as a model protein. In phosphate buffer (PB) the absorption intensity decreased drastically along with pronounced shoulders due to dye aggregation leading to complete fluorescence quenching. However, in the presence of BSA, the intensity of shoulder bands reduced coupled with a sharp increase in the absorption associated with monomeric dye with the increasing concentration of BSA, indicating the prevention of dye aggregation owing to dye-BSA interactions. The prevention of dye aggregation due to dye-BSA interaction also led to a tremendous increase in the fluorescence intensities as a function of the increasing BSA concentrations. Site-selective study off BSA showed that these dyes bind preferably at Site-II using hydrophilic interactions. Thus, these dyes show great potential as FRET or fluorescent on/off probes for biosensing, non-covalent labeling of proteins, and fluorescent probes for bioimaging.