Amine-conjugated FRET coumarin-cyanine ratiometric probes for sensitive viscosity and HSA imaging in live cells and tissues

Document Type

Article

Publication Date

8-15-2026

Department

Department of Chemistry; Department of Biological Sciences; Health Research Institute

Abstract

We designed and synthesized two novel ratiometric near-infrared fluorescent probes, probe A and probe B, tailored for sensitive detection of intracellular viscosity and human serum albumin (HSA) dynamics. Probe A integrates a coumarin donor with a cyanine acceptor to enable ratiometric fluorescence changes upon HSA binding, exhibiting increased visible emission and decreased near-infrared fluorescence. Probe B is engineered with a hemicyanine scaffold that responds to viscosity changes by enhancing visible fluorescence and moderately increasing near-infrared emission, allowing precise ratiometric sensing of microenvironmental viscosity. Both probes show excellent selectivity against biologically relevant amino acids, metal ions, and anions, and exhibit high photostability with less than 3% fluorescence ratio decrease over two hours of continuous excitation. Cytotoxicity assays confirmed low toxicity, with HeLa cell viability remaining above 85% after 48 h at concentrations up to 40 μM, supporting their application for long-term live-cell imaging. Using confocal microscopy, probe B sensitively detected intracellular viscosity increases induced by Monensin, Lipopolysaccharide, and Nystatin treatments in HeLa cells, with ratiometric fluorescence changes correlating with viscosity elevations. Co-localization with mitochondrial dye IR-780 confirmed mitochondrial targeting (Pearson’s coefficient 0.939). Probe B also effectively reported viscosity changes in vivo in Drosophila melanogaster larvae under similar stress conditions. Probe A enabled quantitative ratiometric imaging of HSA dynamics in live cells, with fluorescence changes strongly correlated to albumin concentration. Application of probe A to kidney tissue sections from normal and polycystic kidney disease models revealed significantly elevated fluorescence in diseased tissues, indicating increased intracellular viscosity linked to pathological remodeling. Together, these findings establish probes A and B as versatile, selective, and photostable fluorescent tools for real-time imaging of intracellular viscosity and protein dynamics, with broad potential for biomedical research and disease diagnosis.

Publication Title

Sensors and Actuators B: Chemical

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