基于罗丹明B为荧光基团的三肽荧光探针的设计及应用研究

Design and application of tripeptide fluorescent probe based on rhodamine B as fluorophore

目的:基于铜离子和硫离子对罗丹明B标记的多肽荧光猝灭和荧光恢复作用,建立在活细胞和超纯水中连续检测Cu^(2+)和S^(2-)的方法。方法:采用多肽固相合成技术制备了RhB-Gly-Gly-His,它与Cu^(2+)等量结合形成配合物,该配合物可检测活细胞中的S^(2-)、半胱氨酸(Cys)、谷光甘肽(GSH),同时通过Job曲线和计算机辅助模拟计算预测了该荧光传感器的结构和结合模式。结果:该多肽荧光传感器能与Cu^(2+)等量结合发生荧光淬灭,使用LOD=3σ/k方程计算的荧光滴定测量的检测限为143 nmol·L^(-1)(Cu^(2+))和408 nmol·L^(-1)(S^(2-))。结论:成功地合成了RhB-Gly-Gly-His,该荧光传感器具有荧光发射强、水溶性好、生物相容性好、毒性低等特点。该荧光传感器具有较高的选择性和灵敏度,已成功地用于Cu^(2+)和S^(2-)的定性和定量检测,且拥有良好的选择性,在细胞中的体内成像突出了其在S^(2-),Cys,GSH相关疾病的病理诊断中具有广阔的前景。

Objective:To establish a method for the continuous detection of Cu^(2+)and S^(2-)in living cells and ultrapure water based on the fluorescence bursting and fluorescence recovery effects of Cu^(2+)and S^(2-)on rhodamine B-labeled peptides.Methods:RhB-Gly-Gly-His was prepared by solid-phase peptide synthesis technique.RhB-Gly-Gly-His was equivalently bound with Cu^(2+)to form a complex which could detect S^(2-),Cys,and GSH in living cells.The structure and binding mode of this fluorescent sensor were predicted by Job curve and computer-aided simulation calculation.Results:The peptide fluorescent sensor binds with Cu^(2+)in equal amounts to undergo fluorescence quenching.The detection limits of fluorescence titration measurements were 143 nmol·L^(-1)(Cu^(2+))and 408 nmol·L^(-1)(S^(2-))with the LOD=3σ/k equation.Conclusion:RhB-Gly-GlyHis was successfully synthesized,and the fluorescent sensor has strong fluorescence emission,good water solubility,good biocompatibility and low toxicity.With high selectivity and sensitivity,this fluorescent sensor has been successfully used for the qualitative and quantitative detection of Cu^(2+)and S^(2-).This fluorescent sensor possesses good selectivity for in vivo imaging in cells and highlights a promising future in the pathological diagnosis of S^(2-),Cys,and GSH in related diseases.