The Construction And Biosensing Study Of High-selectivity Fluorescent Probe For Nitrite Oxide

Nitric oxide?NO?,a ubiquitous signaling molecule in biological systems,plays an important role in the cardiovascular,immune,reproductive,central and peripheral nervous systems.The study of the production and action of NO in organisms is essential for understanding the relevant physiological and pathological processes.Among various cellular biology tools,fluorescent probes can monitor active molecules in cells and living organisms in real time,in terms of their convenience,sensitivity,visualization,and noninvasiveness,and become practical tools in biomedical research and disease diagnosis and treatment.Therefore,the development of fluorescent probes that specifically monitor NO in real time under physiological conditions can provide impetus to the development of related biomedical fields.In the past ten years,a large number of NO fluorescent probes have been exploited,which typically include metal-ligand complexe and o-phenylenediamine based ones.However,the metal-ligand complexe-based ones have poor biocompatibility and the o-phenylenediamine-based ones have limitations that need to be addressed,such as the possible interference by dehydroascorbic acid/ascorbic acid/methylglyoxal?DHA/AA/MGO?,unfavorable pH-sensitivity of their fluorescence signals,and relatively slow response rate.Therefore,we are committed to the development of fluorescent probes that can overcome the above-mentioned disadvantages in order to be better able to be used for NO detection in complex biological systems.The main research contents of this paper are as follows:?1?We present two aromatic primary monoamine-based NO fluorescent probes 2.1 and NIR-2.1 by exploring the reductive deamination reaction of the electron-rich p-methoxyaniline group with NO in aerobic condition.The superiority of the two probes could be illustrated by their quick,stable,sensitive,and specific fluorescence off-on responses for NO over a series of biologically relevant interfering species,including reactive oxygen species,DHA/MGO,biothiols,and metal ions.Coupled with the good cell permeability and low cytotoxicity,the two probes have successfully been applied to image the endogenous NO in RAW264.7 macrophages stimulated by LPS/IFN-?.Moreover,the fluorescence response of NIR-2.1 for NO occurs in the physiologically favorable NIR region,enabling its further use to image endogenous NO in an inflamed mouse model.?2?We report a o-phenylenediamine?OPD?-locked Si-rhodamine deoxylactam,i.e.deOxy-DALSiR,as a near-infrared fluorescent probe for selective and sensitive detection of NO in living cells and bodies.Not only could the probe overcome the limitations suffered by the most widely used and commercialized OPD-type fluorescent NO probes,such as possible interferencebydehydroascorbicacid/ascorbicacid/methylglyoxal,pH-sensitive fluorescence output,and short excitation and emission wavelengths,but also avoid the serious interference of cysteine found in the OPD-locked rhodamine lactam-type fluorescent NO probes developed later.What's more,the probe is fairly sensitive to NO,as indicated by the rapid fluorescence response?within seconds?,huge fluorescence off-on ratio?6300-fold?,and ultra-low detection limit?0.12 nM?.Its effectiveness and practicability have been demonstrated by the successful imaging of endogenous NO in RAW 264.7 macrophages,pancreatic?-cells,and endothelial EA.hy926 cells,as well as in the inflamed and diabetic mouse models.The probe was greatly expected to be a useful imaging tool for studying NO-related health and disease.?3?Most NO fluorescent probes sensor NO by detecting N₂O₃-its own oxidation product.However,N₂O₃ does not completely reflect the presence of NO.We have developed a fluorescent NO probe DA which can simultaneously two downstream products of NO metabolism?N₂O₃ and ONOO^-?.Probe DA reacts with N₂O₃ and ONOO^-within seconds,avoid the interference of ROS,DHA/AA/MGO,biothiols,and metal ions.and has a low background fluorescence interference,and the fluorescence increases after the reaction.Doubled,the detection limit was as low as 0.8 nM and 0.14nM.Coupled with the good cell permeability and low cytotoxicity,the two probes have successfully been applied to image the endogenous NO?N₂O₃and ONOO^-?in RAW264.7 macrophages stimulated by LPS/IFN-?,and exogenous NO?N₂O₃ and ONOO^-?in HeLa cells.