Preparation And Application Of Fluorescent Nanoprobes For Simultaneous Recognition Of Phosphorylated Proteins And H₂O₂/H₂S

Active species in living organisms,each performs their own functions and interaction,constitute a complex regulatory network involved in achieving and regulating biological processes.Hydrogen peroxide（H₂O₂）is a key factor in cell signaling that penetrates cell membranes,activates signaling pathways,and participates in biological processes such as immune response,cell growth and proliferation.Hydrogen sulfide（H₂S）is the third largest endogenous gas signaling molecule after carbon monoxide（CO）and nitric oxide（NO）,and plays a crucial role in a series of physiological processes such as stimulating vasodilation,regulating neural excitation,inhibiting inflammatory processes,and maintaining redox homeostasis.Protein phosphorylation is an important post-translational modification involved in signal transduction,gene transcription,apoptosis,metabolism,and almost all other physiological and abnormal pathological processes,and is able to regulate protein function by altering the structure of proteins.Therefore,the development of new analytical methods for the simultaneous identification and detection of metabolic changes in multiple active species and the in-depth study of the correlation between different active species in specific pathological processes of organisms are of great importance for understanding the pathogenesis of diseases.Based on this,metal-organic framework materials（MOFs）with high reactivity centers,high stability,low toxicity and adjustable structure were selected as carriers,and two novel nanofluorescent probes were developed by modifying small molecule fluorescent probes to achieve the simultaneous recognition and imaging of phosphorylated protein and H₂O₂,phosphorylated protein and H₂S,respectively.Finally,the probes were applied to the study of the interrelationship of phosphorylated proteins with H₂O₂,phosphorylated proteins and H₂S in the pathological processes of pneumonia and acute kidney injury（AKI）.The following studies was carried out:1.The nanofluorescent probe UIO-TCPP-NP,which specifically recognizes both H₂O₂ and phosphorylated proteins,was prepared using borate ester and Zr（Ⅳ）as the specific recognition unit and active center,combined with naphthalimide and porphyrin.The addition of H₂O₂ leads to the cleavage of the borate ester,which changes the push-pull electronic effect of the conjugated system and enhances the fluorescence at 550 nm,with a linear relationship at 0-200μM and a correlation coefficient of 0.9924.The addition of phosphate to the solution of UIO-TCPP-NP,due to the specific interaction between Zr（Ⅳ）and the phosphate group,the quenched porphyrin ligand recovers its fluorescence at 650 nm with a linear relationship at 0-200μM and a correlation coefficient of 0.9998.Finally,the UIO-TCPP-NP was successfully applied to in situ fluorescence imaging of the lungs of mice with pneumonia,and the levels of H₂O₂ and phosphorylation during lung inflammation were analyzed to be significantly higher than those of normal mice.This work provides a new analytical approach to study the potential relevance of H₂O₂ and phosphorylated proteins during LPS-induced pneumonia and the mechanisms of disease modulation.2.The nanofluorescent probe UIO-TCPP-NF,which specifically recognizes both H₂S and phosphorylated proteins,was prepared by combining fluorescein and porphyrin with two-photon properties,using dinitrophenyl ether and Zr（Ⅳ）as specific reaction targets and active centers.The probe reacts with H₂S,and the C-O bond breaks,releasing fluorescein and restoring fluorescence at 518 nm with a linear relationship at 0-80μM and a correlation coefficient of0.9884.Upon encountering the phosphate group,the porphyrin fluorescence quenched by charge transfer is restored due to the specific interaction of Zr（Ⅳ）with the phosphate group,and the fluorescence is enhanced at 650 nm with a linear relationship at 0-80μM and a correlation coefficient of 0.9948.In vitro experiments confirmed the good selective recognition ability of the probe.Finally,the probe was applied to the detection and imaging of H₂S and phosphorylated protein in a mouse model of AKI caused by sepsis,and the experimental results showed that the H₂S content decreased and phosphorylated protein level increased.This work provides a new analytical method to study the signaling pathways related to H₂S concentration and phosphorylated protein to reveal the disease mechanism.