A red-emissive tripodal nanoprobe for the discrimination of serum albumin with conformational change from Y- to ω-like and probing mitochondrial viscosity†

Serum albumins, such as bovine serum albumin (BSA) and human serum albumin (HSA), play a crucial role in various biological processes. Discrimination between BSA and HSA is challenging due to their similar structures and reactivity. Here, we report mitochondria-targeted red fluorescent tripodal nanoprobe DMAS-TP, forming spherical nano-aggregates (∼90 nm) in water for discrimination of BSA over HSA and detection and imaging of viscosity in HeLa cells. DMAS-TP exhibits an ∼56-fold fluorescence intensity increase in 95% glycerol compared to water, which indicates restricted movement in high-viscosity solvents. Furthermore, the addition of 5 equiv. BSA and HSA to DMAS-TP solution displays ∼50-fold and ∼10-fold fluorescence intensity increases at 630 nm (λ_ex 490 nm), respectively, and can detect as low as 20 nM BSA and 140 nM HSA. The fluorescence anisotropy plot shows that the DMAS-TP anisotropy values decrease from 0.234 to 0.185 with increasing [BSA/HSA], suggesting a conformational change from Y- to ω-like structure. The fluorescence lifetime of DMAS-TP increases gradually with BSA/HSA, suggesting dynamic complexation. DMAS-TP aggregates diminish to 6 nm particles when encapsulated in BSA confirmed by DLS, SEM, and TEM, with reduced fluorescence intensity in the presence of bilirubin, indicating that DMAS-TP binds within the BSA cavity near site IB. DMAS-TP is highly permeable to HeLa cells and shows a strong affinity for mitochondria, making it suitable for imaging viscosity and BSA via strong fluorescence in the red channel.