Detection Of Nitric Oxide In Cells By Small Molecule Fluorescence Probe

Objective:The content of nitric oxide in cells was detected by the newly developed small molecule fluorescence probe combined with molecular imaging technology,so as to achieve the visual detection of nitric oxide gas in living tissues.Methods:Synthesis of small molecule fluorescent probe: take naphthyl anhydride as fluorescent dye,synthesize small molecule fluorescent probe with Schiff base structure through three steps of reaction,this probe has good specificity for NO.1.Synthesis of small molecule fluorescent probe: Using 4-bromo-1,8-naphthalic anhydride as fluorescent dye,a small molecule fluorescent probe with Schiff base structure was synthesized through a three-step organic reaction,which showed good specificity against NO.2.Performance of small molecule fluorescence probe: The response of the probe to NO was observed by detecting the absorbance and fluorescence intensity of the control group(containing only probe)and the experimental group(containing probe and NO).3.Ion selectivity experiment: The synthesized small molecule fluorescent probe was reacted with other small molecules in the body that may interfere with its selectivity,and the fluorescence intensity after the combination of the small molecule fluorescent probe and interfering ions was observed,thus indicating the specificity of the probe to NO.4.Ion interference experiment: After combining the small molecule fluorescent probe with NO,other interfering molecules are added to observe whether interfering ions affect the fluorescence intensity of the probe combined with NO.5.pH stability: Through the comparison of fluorescence intensity between the control group(containing only probe)and the experimental group(containing probe and NO)under different pH conditions,observe its stability under different pH conditions.6.Cytotoxicity test: Different concentrations of probes were added into the medium to observe the influence of probes on cell activity.7.Cell imaging experiment: Cells were incubated with the probe,and nitric oxide donors and nitric oxide inhibitors with different concentrations were added to observe the changes in fluorescence intensity after the probe was combined with NO in cells.Results:1.The structure of the newly formed small molecule fluorescence probe was analyzed,and the results of mass spectrometry,nuclear magnetic resonance hydrogen spectroscopy and nuclear magnetic resonance carbon spectroscopy confirmed that the newly formed small molecule fluorescence probe was consistent with the designed molecular structure.2.The UV-visible absorption spectra of the control group(containing only the probe)and the experimental group(containing the probe and NO)showed significant displacement fluctuation,and the fluorescence spectra showed that the fluorescence intensity of the experimental group was significantly higher than that of the control group,indicating that the small molecular fluorescence probe was responsive to NO.3.The small molecule fluorescence probe reacts with other small molecules that affect its selectivity.The fluorescence spectra show that the small molecule fluorescence probe has strong fluorescence intensity when it only responds with NO,indicating that the small molecule fluorescence probe has good selectivity for NO.4.The fluorescence intensity increased and remained stable after the response of the small molecule fluorescence probe with NO.When other interfering ions were added,the fluorescence map showed NO significant change in the fluorescence intensity,indicating that the small molecule fluorescence probe had good specificity to NO and strong antiinterference ability.5.The results of PH stability experiment showed that the fluorescence intensity of the fluorescence probe fluctuated little in the range of PH=4-9,and PH had little interference with the fluorescence probe,indicating that the probe had strong stability in the living tissue environment and was suitable for imaging experiments in living tissue.6.Cell survival rate results showed that high concentration of fluorescent probe had little effect on cell survival rate,indicating that the probe was suitable for application in cell imaging experiments.7.Cell imaging experiment results showed that in living cells,fluorescence intensity was significantly enhanced after the probe was combined with NO,and decreased after the addition of NO inhibitor.Conclusion:1.The small molecule fluorescence probe designed in this paper contains Schiff base structure,which can bind specifically to NO.The fluorescence intensity of the fluorescence probe is weak,but the fluorescence intensity is enhanced after binding with NO.2.The fluorescence probe has good selectivity and specificity,which is suitable for the detection of NO.3.This fluorescent probe has little damage to living cells and can be used for the detection of NO in living cells.4.The fluorescence probe showed obvious imaging in living cells and was suitable for visual detection of NO in vivo and in vitro.5.As a signaling molecule,the content of NO increases in inflammatory tissues and some diseases.The visual detection of NO by using the probe designed in this paper is conducive to monitoring the transfer of signal molecules in basic research,monitoring the progress of inflammatory response,and assisting the diagnosis of some diseases.