基于核酸适配体和G-四链体免标记荧光分析法检测氯霉素

Detection of chloramphenicol based on nucleic acid aptamer and G-quadruplex labeling free fluorescence analysis

中文关键词:  氯霉素  适配体  G-四链体  N-甲基卟啉二丙酸IX

英文关键词:chloramphenicol  aptamer  G-quadruplex  N-methylmesoporphyrin IX

中文摘要:

      目的 基于核酸适配体和G-四链体(G-quadruplex, G4)结构建立一种免标记荧光分析法检测氯霉素(chloramphenicol, CAP)含量, 应用于化学分析、环境检测、食品安全等领域。方法 G4探针和适配体(aptamer, AP)在Tris-HCl缓冲液中合成, 由于G4探针与适配体的结合抑制了G4的形成, N-甲基卟啉二丙酸IX (N-methylmesoporphyrin IX, NMM)染料无法被G4结构增强, 荧光强度较弱, 加入CAP竞争结合适配体, 使得G4探针结构改变, 通过荧光强度产生的变化分析样品中的CAP含量。围绕G4检测探针进行系列优化, 包括探针序列、Tris-HCl缓冲液体系中钾离子和镁离子浓度、G4探针浓度与AP浓度比例。结果 当CAP质量浓度在1~10 ng/mL时, 体系的荧光强度增长值(Y)与CAP质量浓度(X)呈线性关系, 线性方程为Y=59.091X+1.733 (r2=0.9939)。根据3倍相对标准偏差(3σ/K, n=11)计算该方法检出限, 为0.518 ng/mL。本方法对卡那霉素、链霉素和庆大霉素无荧光响应, 具有良好选择性和特异性。结论 本方法重复性和实用性好, 可应用于化学分析、环境检测、食品安全等领域。

英文摘要:

      Objective To establish a labeling free fluorescence analysis method to detect chloramphenicol (CAP) content based on the nucleic acid aptamer and G-quadruplex (G4) structure, and apply it to chemical analysis, environmental detection, food safety and other fields. Methods G4 probe and aptamer (AP) were synthesized in Tris-HCl buffer solution, as the combination of G4 probe and aptamer inhibited the formation of G4, N-methylmesoporphyrin IX (NMM) dye could not be enhanced by the G4 structure with weak fluorescence intensity, CAP was added to compete for the binding aptamer, so that the structure of G4 probe was changed, and the content of chloramphenicol in the sample was analyzed by the change in fluorescence intensity. A series of optimizations were performed around the G4 detection probe, including the probe sequence, the concentration of potassium and magnesium ions in the Tris-HCl buffer liquid system and the ratio of the G4 probe concentration to the AP concentration. Results The fluorescence intensity growth value (Y) of the system showed a linear relationship with the CAP mass concentration (X) when the mass concentration of chloramphenicol was in the range of 1?10 ng/mL, and the linear equation was Y=59.091X+1.733 (r2=0.9939). The limit of detection for the method were calculated as 0.518 ng/mL based on 3 times the relative standard deviation (3σ/K, n=11). The method had no fluorescent response to kanamycin, streptomycin and gentamicin, and had good selectivity and specificity. Conclusion This method has good repeatability and practicability, and can be applied to chemical analysis, environmental detection, food safety and other fields.