基于四甲基联苯胺和吖啶橙间荧光共振能量转移的比率型荧光测定左氧氟沙星

Ratiometric Fluorescence Determination of Levofloxacin Based on Fluorescence Resonance Energy Transfer Between Tetramethylbenzidine and Acridine Orange

基于3,3’,5,5’(四甲基联苯胺(3,3’,5,5’-tetramethylbenzidine,TMB)和吖啶橙(AO)之间荧光共振能量转移(FRET),建立了一种快速、低背景干扰、高灵敏度测定盐酸左氧氟沙星(LVF)的新型比率型荧光探针。在pH 5.0 NaAc-HCl缓冲溶液中,在310 nm的光激发下,TMB在350~500 nm处的荧光光谱和AO的吸收光谱重叠。以TMB作为能量供体,AO作为能量受体,构建了FRET体系。根据能量转移理论,该体系的荧光共振能量转移效率为62.5%,供体-受体间距离为2.17 nm,进一步说明TMB和AO之间发生了FRET。当在体系中加入LVF后,TMB将荧光能量转移给LVF,LVF又作为供体将能量转移给AO。LVF在TMB和AO之间起到桥梁作用,LVF将吸收的TMB荧光能量转移给AO,使得TMB荧光强度明显降低,AO的荧光强度则显著增加,从而提高了体系的FRET效率。在最优实验条件下,F546 nm与F402 nm之比与LVF浓度(2~80μmol·L^(-1))之间存在良好的线性关系,线性回归方程为F_(546 nm)/F_(402 nm)=87.916c+3.108,线性相关系数为0.9993,检出限(LOD)为15.7 nmol·L^(-1)。一些常见的阳离子(K^(+),Mg^(2+),Ca^(2+),Cu^(2+),Mn^(2+),Zn^(2+),Co^(2+),Ni^(2+),Cr^(3+)等)、阴离子(F^(-),Br^(-),NO_(3)^(-),IO_(3)^(-),CO_(3)^(2-),SO_(4)^(2-)等)、糖类(葡萄糖,蔗糖和淀粉)、药物(谷胱甘肽,抗坏血酸和异烟肼)和几种氨基酸(甘氨酸,亮氨酸,半胱氨酸等)均不干扰LVF的测定,表明该比率型荧光探针对LVF具有高选择性。该方法用于商用药物制剂中LVF含量的测定,加标回收率在93%~97%之间。该比率型荧光探针在临床研究中对LVF的检测具有较大的应用潜力,为开发一种简便、选择性和灵敏的检测药物制剂中LVF含量的传感器提供了较好的理论依据,同时为提高LVF临床用药的安全性与合理性水平提供了一定的方法。

A novel ratiometric fluorescence probe for the determination of levofloxacin hydrochloride(LVF)was established based on fluorescence resonance energy transfer(FRET)between 3,3’,5,5’-tetramethylbenzidine(TMB)and acridine orange(AO).Under 310 nm excitation,the fluorescence spectra of TMB at 350~500 nm overlap with the absorption spectra of AO in the pH 5.0 NaAc-HCl buffer solution.Accordingly,the FRET system is constructed with TMB as the energy donor and AO as the energy receptor.According to the energy transfer theory,the FRET efficiency of the system is 62.5%,and the distance between the donor and receptors is 2.17 nm,further indicating that FRET occurs between TMB and AO.When LVF is added,TMB transfers the fluorescence energy to LVF,which transfers the energy to AO as a donor.LVF acts as a bridge between TMB and AO.LVF transfers the absorbed fluorescence energy of TMB to AO,resulting in a significant decrease in the fluorescence intensity of TMB and a significant increase in the fluorescence intensity of AO,thus improving the FRET efficiency of the system.Under the optimal experimental conditions,there is a good linear relationship between the ratio of F546 nm to F402 nm and LVF concentration in the 2~80μmol·L^(-1) range.The linear regression equation is F_(546 nm)/F_(402 nm)=87.916c+3.108,the linear correlation coefficient is 0.9993,and the detection limit(LOD)is 15.7 nmol·L^(-1).Some common cations(K^(+),Mg^(2+),Ca^(2+),Cu^(2+),Mn^(2+),Zn^(2+),Co^(2+),Ni^(2+),Cr^(3+),etc),anions(F-,Br-,NO-3,IO-3,CO_(2)-3,SO_(2)-4,etc),sugars(glucose,sucrose and starch),drugs(glutathione,ascorbic acid and isoniazid)and several amino acids(glycine,leucine,cysteine,etc)do not interfere with the determination of levofloxacin,indicating that the ratiometric fluorescent probe has high selectivity for LVF.The method was applied to determine LVF in commercial pharmaceutical preparations with a recovery of 93%~97%.The ratiometric fluorescence probe has great potential in clinical application for the detection of LVF,which provides a good theoretical basis for the development of a simple,selective and sensitive sensor for the determination of LVF in pharmaceutical preparations,and a certain method guidance for improving the safety and rationality of LVF inclinical medication.