Design,Synthesis And Application Of Nitroreductase And Nitric Oxide Fluorescent Probes

Objectives:Early diagnosis and treatment of diseases have always been at the center and the challenge of biomedical research.According to the Global Oral Health Report released by the World Health Organization in 2022,about 3.5 billion people worldwide have oral diseases,of which oral cancer is the most malignant oral disease.Tissue biopsy is the gold standard of oral cancer diagnosis,but invasive screening technology is laborious,time-consuming and traumatic.Fluorescent probes have the advantages of high sensitivity,rapid response and simple technology,and are expected to solve some problems in early non-invasive diagnosis of oral diseases to a certain extent.Therefore,in this study,oral diseases were taken as the main research objective,two new fluorescent probes were designed and synthesized as analytical tools,and their applications in the detection and imaging of disease markers nitroreductase（NTR）and nitric oxide（NO）were explored.Methods:An Off-On type fluorescent probe Rhod-NO₂ with Rhodolo derivative structure was designed and synthesized.The structure of the probe was characterized by 1H-NMR,13C-NMR and MS.The spectral properties,selectivity and stability of the probe were systematically studied,and an analytical method for the determination of NTR was established.It was applied to the real-time monitoring of NTR during the growth of Escherichia coli.The cytotoxicity of the probe was investigated by the MTT method,and it was applied to the visualization analysis of NTR concentration in oral cancer cells（Cal-27）under hypoxia.An Off-On near-infrared fluorescence probe CS-Se was designed and synthesized.The structure of the probe was characterized by 1H-NMR and MS.The spectral performance of the probe was systematically studied,and its selectivity and stability were researched.An analytical method for the determination of NO was established.The reaction mechanism of CS-Se and NO was studied by MS and theoretical calculation.It was used to monitor the concentration of NO in the saliva of patients with oral diseases.The cytotoxicity of the probe was investigated by the MTT method,and it was applied to the visual analysis of NO concentration in Cal-27 cells.Results:1、The Rhod-NO₂ fluorescent probe was successfully synthesized,which exhibited high selectivity,high sensitivity（detection limit 0.6ng/m L）,and fast response（30 min）performance for NTR.The Rhod-NO₂ fluorescent probe was successfully synthesized,which exhibited high selectivity,high sensitivity（detection limit,0.6 ng/m L）,and fast response（30 min）performance for NTR.The reaction mechanism is that Rhod-NO₂ generates the amino product Rhod-NH2 under the action of NTR,and the effect of intramolecular charge transfer（ICT）is enhanced to realize the function of fluorescence signal Off-On.The experiment of Escherichia coli showed that Rhod-NO₂ could be used to monitor NTR produced during the growth of Escherichia coli in real-time.Cell experiments showed that Rhod-NO₂ had low toxicity and could be used for visualization analysis of NTR in Cal-27 cells.2、CS-Se fluorescent probe was successfully synthesized,which showed high selectivity,high sensitivity（detection limit,28 n M）and fast response（5 min）to NO.The reaction mechanism is predicted as two processes:（1）NO interacts with Se to form an unstable intermediate CS-Se-NO,and then the CS-Se bond is hydrolyzed to CS;（2）The unstable intermediate CS-Se-NO undergoes a complex coupling reaction to form CS-Se polymer.As NO induces the inner spiral ring of CS-Se to open,the fluorescence signal Off On function is realized.The saliva sample experiment shows that the concentration of NO in the saliva of healthy people is 24-44 μ M.The concentration of the NO in the saliva of patients with oral diseases was conspicuously higher than that of healthy people,and there was a huge difference among healthy individuals,periodontitis individuals and oral cancer individuals.Cell experiments showed that CS-Se had low toxicity and could be used for visual analysis of NO in Cal-27 cells.Conclusions:1.Rhod-NO₂,a fluorescent probe for detecting NTR,has been developed,which may become a microbial growth indicator tool and provide a new analytical tool for further understanding the biological function of NTR in the process of oral diseases.2.A near-infrared fluorescence probe CS-Se has been developed to detect NO,which reveals the relationship between the concentration of NO in saliva and oral diseases,and provides a new analytical tool for the research and early diagnosis of NO in oral diseases.