Development of D–π–A hemicyanine based NIR fluorescent probes for sensitive detection of intracellular viscosity variations

Abstract

Intracellular viscosity plays a crucial role in regulating cellular signaling and molecular transport. Abnormal variations in viscosity have been associated with various diseases, particularly cancer. Traditional viscometers are inadequate for measuring intracellular viscosity, prompting the development of alternative sensing methods. In this work, two small-molecule fluorescent probes were designed and compared in terms of their responses to viscosity variations. These probes possess a donor–π–acceptor (D–π–A) structure, where the 4-(dimethylamino)phenyl group serves as the electron donor, while two different electron-accepting groups are incorporated. Furthermore, two π-bonds were introduced to enhance fluorescence efficiency within the far-red to near-infrared (NIR) region (λ = 650–900 nm), enabling deeper tissue penetration. In this work, the fluorescent probes, NIR-IND and NIR-BZO, were successfully prepared via a few-step synthesis. They exhibited moderate polarity sensitivity, pronounced viscosity-responsive NIR fluorescence properties, large Stokes shift (up to 162 nm), a broad pH stability range (3–13), high specificity, and strong photostability. The fluorescence intensities of NIR-IND and NIR-BZO at 690 and 688 nm, respectively, increased by to 43-fold from pure water (0.75 cP) to pure glycerol (630.4 cP). In addition, computational studies strongly supported the designation of these probes as effective viscosity-responsive fluorophores, highlighting their potential for applications in viscosity sensing and intracellular imaging, rather than polarity detection.