Synthesis of polar-insensitive phenothiazine-based fluorescent probes for viscosity detection in mitochondria

Mitochondria, the primary energy-producing organelles, critically regulate cellular activities. Their internal viscosity—a key microenvironmental parameter—directly affects material transport and energy conversion, with aberrant levels linked to disease pathogenesis. Most reported mitochondrial viscosity probes were susceptible to polar interference, resulting in insufficient specificity for viscosity detection. This study designed and developed a novel polar-insensitive fluorescent probe, Y-CN , based on a phenothiazine skeleton - a small molecular compound with excellent fluorescent properties. The experimental data revealed an excellent linear relationship (R 2  > 0.99) between Y-CN 's fluorescence intensity and viscosity over a wide range from 1.005 to 219 cP. When viscosity increased from 1.005 cP (aqueous buffer) to 219 cP, Y-CN demonstrated a dramatic 209-fold fluorescence amplification. Furthermore, the probe exhibited excellent selectivity, pH stability, and low toxicity. Moreover, Y-CN could detect viscosity changes induced by food thickeners. Y-CN successfully monitored dynamic changes in mitochondrial viscosity within live cells, providing a powerful tool for specific investigation of mitochondria-associated physiological and pathological processes, with significant potential for biological applications.