Construction And Cell Imaging Of Viscosity And Peroxynitrite Fluorescent Probe

Viscosity is an important parameter of the intracellular microenvironment and plays a crucial role in controlling the physical or chemical behavior of local molecules.Abnormal viscosity levels can cause cellular pathologies leading to the development of various diseases.Peroxynitrite(ONOO~-),a type of reactive oxygenated nitrogen species(RONS),is a strong oxidizing and nucleophilic agent.Normal levels of peroxynitrite can promote intracellular signaling,but excessive levels of peroxynitrite can lead to damage to various intracellular organelles,leading to various diseases.The ability to detect changes in intracellular viscosity and peroxynitrite levels in real time is therefore important for the prevention and treatment of related diseases.Fluorescent probe imaging is currently one of the most powerful techniques available for the continuous observation of intracellular processes in living cells and tissues,offering the advantages of high spatial and temporal resolution,in situ monitoring and real-time visualization.To date,many fluorescent probes have been developed that can track in real time the levels of various parameters in the internal environment including viscosity and polarity and the levels of various substances including peroxynitrite,sulfur dioxide and hypochlorous acid.However,the emission wavelengths and Stokes shifts of most fluorescent probes are not at the desired level,resulting in high interference from background fluorescence during testing and damage to cells,etc.Therefore,there is an urgent need to develop a new type of NIR fluorescent probe with a large Stokes shift.Based on the above research status and problems encountered with viscosity fluorescent probes and peroxynitrite fluorescent probes,two fluorescent probes that can be used to specifically detect intracellular viscosity levels and one fluorescent probe that can be used to detect intracellular peroxynitrite content have been designed and their spectral properties and cell imaging results have been analyzed and investigated.First,a near-infrared fluorescent probe with a large Stokes shift was synthesized for the detection of viscosity in the intracellular environment using dicyanoisophorone as the fluorophore and phenothiazine structure as the conjugate group.The spectroscopic properties show that the probe is sensitive and responds rapidly to viscosity in the environment and emits fluorescence at 725 nm with good photo stability at an excitation wavelength of 500 nm.Cellular tests show that the probe responds promptly to changes in viscosity in the intracellular environment and has low cytotoxicity.Second,a new viscosity fluorescent probe was designed and synthesized using the selenobenzopyran structure as the base fluorophore and 4–(diethylamino)salicylaldehyde as the molecular rotor,with a further red-shift in emission wavelength to 750 nm.Spectroscopic tests showed that the probe was sensitive to environmental viscosity and had good stability.Further cellular testing revealed that the probe can rapidly respond to changes in viscosity in cells.Third,a near-infrared large Stokes shift fluorescent probe for the specific detection of peroxynitrite was designed using a selenobenzopyran structure as the base fluorophore and a diphenylphosphate group as the specific recognition group for peroxynitrite.Spectroscopic results show that the probe specifically responds to peroxynitrite and emits fluorescence at a wavelength of 770 nm at an excitation wavelength of 600 nm.Cellular tests demonstrate that the probe specifically responds to intracellular peroxynitrite.