Fluorescence Detection Technique For ONOO~- And Cysteine In Diabetic Encephalopathy Injury

Objective To detect changes in peroxynitrite(ONOO~-)in diabetic encephalopathy injury by designing and synthesizing a specific small molecule fluorescent probe NP and a proportional two-photon fluorescent probe Ro-Np based on NP and rhodamine.Using the structure of Azamonardine(Aza)with high quantum yield as the parent,we designed and synthesized the fluorescent probe Aza-acryl to detect the changes of Cysteine(Cys)in diabetic encephalopathy injury,providing a new technical tool for the detection of diabetic encephalopathy injury.Methods A specific ONOO~-fluorescent probe NP was designed and synthesized based on 1,8-naphthalimide as a two-photon fluorescent group for fluorescent probes and 4-nitrophenyl pyruvate with fast and sensitive reaction as a leaving group for fluorescent probes,and a proportional twophoton fluorescent probe Ro-Np based on np and rhodamine was designed and synthesized based on this.Aza-acryl was designed and synthesized based on the structure of Azamonardine(Aza)with high quantum yield,and a fluorescent probe for the detection of Cys in biothiols was synthesized.change,selectivity experiments to detect the selectivity of the probe to the reactants in various amino acids,metal ions,interference experiments to detect the fluorescence recovery interference of the probe to the reactants on the basis of various amino acids,metal ions.Fluorescence imaging of exogenous and endogenous generation of reactants in cells by drug intervention.Fluorescence imaging of zebrafish by drug intervention with a fluorescent probe.The brains of mice with diabetic encephalopathy were fluorescently imaged with a fluorescent probe by establishing a mouse model of diabetic encephalopathy.Results In the probe characterization experiments,compared with the blank group,the fluorescent probe NP and probe Ro-Np showed high intensity of fluorescence response to ONOO~-,short reaction time,both completed within1 min and stable fluorescence recovery changes at physiological p H 7.4,without interference from other amino acids and metal ions,selective and specific for ONOO~-pairs.In the fluorescence imaging experiments of exogenous and endogenous production of ONOO~-in cells,the fluorescence of probe NP and probe Ro-Np was gradually enhanced with increasing concentrations of SIN-1 and LPS in the SIN-1 and LPS groups compared with the control group.In the fluorescence imaging experiment in zebrafish,the fluorescence of probe NP was significantly enhanced in the SIN-1 group compared with the control group.In fluorescence imaging experiments in diabetic encephalopathy mice,the fluorescence of probe NP and probe RoNp was significantly enhanced in the diabetic group compared with the control group.Compared with the blank group,the probe Aza-acryl showed high fluorescence response intensity to Cys,and the response reached the most intense value of fluorescence at 5 min and was at the plateau.The fluorescence recovery change was stable under physiological p H 7.4environment,and was not interfered by other amino acids,metal ions,and was selective and specific for Cys pairs.In the fluorescence imaging experiments of cellular exogenous and endogenous production of Cys,the fluorescence of the probe Aza-acryl was significantly weaker in the NEM group compared to the control and Cys groups.In the fluorescence imaging experiments of zebrafish,the fluorescence of the probe Aza-acryl was significantly diminished in the NEM group compared to the control and Cys groups.Conclusions The results show that the three specific small molecule fluorescent probes designed and synthesized successfully detected peroxynitrite(ONOO~-)and biothiol Cys changes in biological systems,thus enabling the detection of peroxynitrite(ONOO~-)and biothiol Cys changes in diabetic encephalopathy injury.