山柰酚及其环糊精包合物作为荧光探针对铜离子识别作用的研究

Fluorescent properties and application for recognizing copper ions based on kaempferol and kaempferol⁃cyclodextrin inclusion

【目的】山柰酚(kaempferol,Kae)是一种广泛存在于植物中的黄酮类化合物,分析Kae的荧光性能,揭示Kae作为荧光探针识别金属离子的机理,为进一步开发黄酮资源提供参考。【方法】实验通过调节pH、制备环糊精包合物等方法,优化Kae的荧光发射条件,探索Kae荧光性能及其应用。【结果】Kae在CH_(3)OH⁃PBS缓冲溶液(体积比1∶99,pH为7.40)中具有荧光发射现象,但荧光强度较弱、稳定性较差。与2⁃羟丙基⁃β⁃环糊精[(2⁃hydroxypropyl)⁃β⁃cyclodextrin,CD]形成包合物后,包合物(Kae⁃CD)的最大荧光发射峰由538 nm红移至552 nm,Stokes位移增大;同时,荧光强度提高,荧光稳定性显著提高,Kae的荧光性能得到明显改善。Kae可以作为荧光探针对水溶液中的铜离子进行定量检测,定量方程为y=-10.61 x+225.8(R^(2)=0.998),线性范围为1.0×10^(-8)~1.7×10^(-6)mol/L,检测限可达4.2×10^(-9)mol/L。但是Fe^(2+)对Kae检测铜离子的过程有一定的干扰,而Kae⁃CD可以有效避免Fe^(2+)的干扰,对铜离子具有更高的选择性,定量方程为y=-8.54 x+708.55(R^(2)=0.997),线性范围为5.0×10^(-8)~5.0×10^(-6)mol/L,检测限可达1.5×10^(-8)mol/L。紫外⁃可见光吸收光谱、Job曲线、红外光谱等研究表明,Kae和Kae⁃CD与铜离子形成化学计量比为2∶1的配合物,促使Kae分子发生分子内电荷转移,从而引起荧光猝灭。【结论】Kae⁃CD比Kae的荧光性能更加优越,二者均可以作为荧光探针对河水中微量铜离子进行检测,检测结果与ICP⁃MS或ICP⁃OES法相比,结果更准确、稳定。

【Objective】Kaempferol(Kae)is a flavonoid that is widely present in plants.By investigating the fluorescence properties and applications of Kae,this study seeks to reveal the recognition mechanisms of metal ions and provide a basis for the development of flavonoid resources.【Method】Kae was extracted from ginkgo leaves.The experimental conditions for obtaining the fluorescence emission spectra of Kae were optimized by adjusting the pH and preparing the inclusion complex with(2⁃hydroxypropyl)⁃β⁃cyclodextrin(CD).The responses of Kae and Kae⁃CD on different metal ions were explored by studying the change of fluorescence.【Result】Interestingly,the fluorescent emission of Kae was observed in the CH_(3)OH⁃PBS buffer solution(volume ratio 1∶99,pH 7.40),however,the fluorescence intensity and stability were poor.When the inclusion complex was formed with Kae and CD,the maximum emission peak shifted from 538 nm to 552 nm,and the Stokes shift increased.Moreover,the fluorescence intensity was greater than that of Kae and more stable.The results of selectivity experiments indicated that fluorescence of Kae was quenched after adding Cu^(2+),fluorescence intensity of Kae decreased in some degree after adding Fe^(2+).And other metal ions had no obvious influence on fluorescence intensity of Kae.Fluorescence titration experiments demonstrated fluorescence intensity of Kae was inversely proportional to Cu^(2+)concentrations.The calibration curve between fluorescence intensity(y)and Cu^(2+)concentration(x)was defined as y=-10.61x+225.8(R^(2)=0.998)with a linear range from 1.0×10^(-8)to 1.7×10^(-6)mol/L.The detection limit was estimated to be 4.2×10^(-9)mol/L.In the Kae⁃CD solution,fluorescence quenching occurred after adding Cu^(2+),and the other metal ions had no obvious effect on fluorescence intensity,which indicated that the Kae⁃CD solution had a higher selectivity for Cu^(2+).Fluorescence titration experiments demonstrated that the linear relationship between fluorescence intensity(y)of Kae⁃CD solution and Cu^(2+)concentration(x)was defined as y=-8.54 x+708.55(R^(2)=0.997)with a linear range from 5.0×10^(-8)to 5.0×10^(-6)mol/L and detection limit was 1.5×10^(-8)mol/L.UV⁃Vis spectra,FT⁃IR spectra,and Job􀆳s plots were used to characterize the mechanisms of Cu^(2+)recognition.These experiments demonstrated that Kae and Kae⁃CD formed complexes with Cu^(2+)in a stoichiometric ratio of 2∶1.When the complex was formed,intramolecular charge transfer(ICT)occurred due to the extension of the conjugated system,thereby causing fluorescence quenching.【Conclusion】As shown by the present study,this method can be successfully applied to determine the concentration of Cu^(2+)in rivers or lakes.Compared with the ICP⁃MS or ICP⁃OES methods,the results obtained using Kae and Kae⁃CD were more accurate and stable,thereby providing a novel,fast and convenient method to quantify Cu^(2+)presence.