| Objective: Hepatocellular carcinoma is the sixth most common cancer in the world and the third leading cause of cancer-related mortality.As the most common type of primary liver cancer,there has been a lot of attention and focus on its diagnosis and treatment.As a major antioxidant,glutathione(GSH)plays an important role in protecting cells from oxidative damage caused by hepatocellular carcinoma.In addition to glutathione(GSH),which is the most abundant biothiol in human body,two other biothiols,cysteine(Cys)and homocysteine(Hcy),are also present in human body,but the structural similarity of these three biothiols makes it difficult to detect all three selectively.In recent years,fluorescent molecular probe assays developed based on coumarins and their derivatives have attracted widespread attention because of their high selectivity,sensitivity,visualization,high biocompatibility,high quantum yield,low detection limit,simple operation and low cost,and have become one of the important tools for biological analysis,organic synthesis and metabolism,and clinical diagnosis.In order to selectively detect GSH from GSH,Cys and Hcy,our team synthesized a new "turn-on" fluorescent probe based on coumarin.Methods: We developed the probe 4-BrCP with coumarin as the fluorescence core and examined a series of properties such as selectivity,sensitivity,minimum detection limit,anti-interference performance,p H range,and photostability by a series of spectroscopic measurements,followed by a cytotoxicity(CCK-8)assay to verify the cytotoxicity of 4-BrCP.The fluorescence imaging performance of 4-BrCP in live cells and organisms and the biological application value of 4-BrCP were verified by live Hep G2 cells as well as C.elegans and zebrafish subjects.Results: The quantum yield of 4-BrCP was calculated as 0.84.After a series of fluorescence spectroscopy experiments,it was demonstrated that4-BrCP could selectively react with GSH in the coexistence of GSH,Cys and Hcy,and its maximum fluorescence emission intensity was at λ=495 nm.After the reaction started,the growth rate of peak emission intensity began to slow down significantly after about 12 min,and from about 23 min,its fluorescence intensity began to stabilize gradually and reached a plateau.The minimum detection limit of 4-BrCP was found to be 9.8 n M by GSH titration,and the probe was stable in the physiological p H range of 5-8 with GSH,and demonstrated good photostability even in different p H environments.In addition,the anti-interference test demonstrated that the probe can detect and react with GSH with high selectivity in complex biological systems without being affected by other common interfering factors.Finally,the low cytotoxicity of 4-BrCP was successfully verified by CCK-8 assay,and the biofluorescence imaging capability of 4-BrCP was confirmed by fluorescence imaging assays on live Hep G2 cells,as well as C.elegans and zebrafish.Conclusion: We have designed and synthesized a novel "turn-on" coumarin fluorescent probe 4-BrCP,which has a series of features such as high selectivity,excellent sensitivity,outstanding anti-interference performance,good photostability,low cytotoxicity,and ideal biofluorescence imaging effect.In view of the above-mentioned good properties of the probe 4-BrCP,we believe that 4-BrCP will greatly facilitate the investigation of early diagnostic screening or basic research of GSH-related diseases including hepatocellular liver cancer,better apply to the sensing monitoring of disease prognosis and diagnosis-related biomarkers including hepatocellular liver cancer,and further design and develop a fluorescent probe drug with 4-BrCP as a scaffold targeted delivery system. |
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