| Hypochlorous acid（HOCl）,as one of the important reactive oxygen species in physiological system,it can maintain the balance of intracellular redox balance and has antibacterial and bactericidal immune effect.However,overexpression of HOCl in the body may cause a series of diseases,such as rheumatoid arthritis and atherosclerosis.A simple,fast and efficient method for the accurate detection of HOCl in physiological systems is of great value to the monitoring of related pathophysiological processes.Among all kinds of detection methods,fluorescence analysis method has been favored by the majority of researchers due to its advantages of high sensitivity,good selectivity,fast response and real-time monitoring.Phenothiazine is a heterocyclic compound with non-planar“butterfly”structure,which has the advantages of easy functionalization and large Stokes shift as a fluorophore parent nucleus.In this article,we successfully designed and synthesized a series of fluorescence probes based on the reaction mechanism that sulfur atoms in phenothiazine nucleus are easily oxidized to sulfoxide（S=O）by HOCl to achieve imaging analysis in cells,tissues and mice.First,a simple structural modification of the reported HOCl probe 1-1 with a short emission wavelength was carried out,and the cyanopyridine salt was introduced into the phenothiazine structure by amplifying the intramolecular charge transfer（ICT）strategy,two long-wavelength emission HOCl fluorescence probes Cl O-1 and Cl O-2 were designed and synthesized.Under the addition of HOCl,these two probes exhibited461-fold and 472-fold fluorescence enhancement at 635 nm and 614 nm,respectively.Among them,compared with Cl O-1 with fast response（5 s）and high sensitivity（DL=1.12 n M）,the probe Cl O-2 with methoxy group has shorter emission wavelength and is more interfered by ONOO-.Therefore,using the probe Cl O-1,lipopolysaccharide（LPS）-induced HOCl overexpression in RAW264.7 cells was achieved,and the up-regulation of HOCl concentration in MCF-7 cells was realized stimulated by 5-FU.Traditional phenothiazine derived HOCl fluorescent probes are not only easy to oxidize sulfur atom,but also have poor chemical stability of active C=C bond,which interferes with selectivity.To further improve the stability and selectivity of the probe,we cycled phenothiazine and coumarin to obtain phenothiazine-fused coumarin PDC with a single sulfur atom reaction site.PDC showed high selectivity and sensitivity to HOCl fluorescence enhancement at the emission wavelength at 503 nm.In addition,we successfully applied probe PDC to image analysis of endogenous HOCl in RAW264.7cells stimulated by multiple inflammatory factors,and effectively evaluated the efficacy of two anti-inflammatory drugs,selenocysteine and methotrexate.The probe was also successfully used to in vivo image changes in HOCl levels by constructing a mouse osteoarthritis model.On the basis of ensuring the high selectivity of phenothiazine-coumarin-based probes,in order to increase their imaging utility,a lysosomal targeting HOCl fluorescent probe PC-Py was successfully synthesized by replacing the carboxyl group in PDC with a positively charged pyridinium salt with stronger electron-withdrawing ability.Compared with PDC,the probe has a longer emission wavelength at 570 nm and a red shift of 67 nm after the interaction with HOCl.The probe has rapid response,good selectivity and a wide range of p H detection.Due to the hypersensitivity of the probe PC-Py（DL=970 p M）,the imaging analysis of HOCl in cancer cells was realized and the effective differentiation between normal cells and cancer cells was achieved.Finally,the co-localization experiment confirmed that the probe could be successfully targeted to lysosomes.In addition to oxidative stress,a particular pathophysiological process is often accompanied by other microenvironmental changes,such as viscosity.Therefore,the simultaneous detection of multiple microenvironmental changes can achieve more accurate detection of related pathophysiological processes.A dual-responsive fluorescent probe PCV was designed and synthesized by introducing a viscosity-responsive molecular rotor into the phenothiazine structure.The probe can display enhanced fluorescence signals for HOCl and viscosity in different fluorescence channels,respectively.With the increase of the viscosity of the system,the probe showed a 5.5-fold fluorescence intensity change in the near-infrared region（710 nm）;with the increase of the HOCl concentration,an 84-fold fluorescence enhancement appeared at 593 nm.In different polar solvents,the probe showed weak fluorescence,eliminating the effect of polarity.The probe has good biocompatibility and has been successfully applied to bioimaging the upregulation of HOCl level or viscosity in cells stimulated by LPS or nystatin.Finally,by constructing a foam cell model,the probe PCV was applied to the simultaneous imaging of changes in HOCl and viscosity levels in early atherosclerosis.At present,some biological experiments are still being carried out and perfected. |
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