基于核酸适配体的荧光法检测中药中赭曲霉素A  

Aptamer-based fluorescent assay for determination of ochratoxin A in herbal medicine

作　　者：秦美君 郭丽敏 耿海琴 李赟 QIN Meijun;GUO Limin;GENG Haiqin;Li Yun(College of Traditional Chinese Medicine and Food Engineering,Shanxi University of Chinese Medicine,Jinzhong 030619)

机构地区：[1]山西中医药大学中药与食品工程学院,晋中030619

出　　处：《分析试验室》2021年第12期1370-1375,共6页Chinese Journal of Analysis Laboratory

基　　金：山西省自然科学基金(201801D221072);国家自然科学基金(81803725)项目资助。

摘　　要：赭曲霉素A(OTA)是污染中药材的重要真菌毒素,严重影响中药材质量和用药安全。在小檗碱溶液中加入OTA和其核酸适配体后,处于随意卷曲状态的核酸适配体会被OTA诱导折叠成为G-四链体构象,使小檗碱的微环境发生改变,从而增强其荧光信号。基于此,本文以OTA核酸适配体为识别原件,小檗碱为荧光探针发展了一种无标记的荧光体系检测OTA。对主要影响因素,包括K^(+), Mg^(2+),小檗碱和核酸适配体浓度进行了优化。在最佳实验条件下,小檗碱荧光信号变化值与OTA浓度在5~200 nmol/L范围内成正比,检出限5 nmol/L。该方法仅使用无标记的核酸适配体完成了OTA的检测,避免了对核酸适配体的繁琐设计和标记。方法有较高的特异性,并成功应用于中药桔梗中OTA的检测,回收率在86.3%~105.6%之间。Ochratoxin A(OTA) is an important mycotoxin that contaminates merbal medicines, which seriously affects the quality and safety of herbal medicines. After adding OTA and aptamer into berberine solution, the aptamer in random curl state will be induced to fold into G-quadruplex conformation by OTA, which will change the microenvironment of berberine and enhance the fluorescence signal of berberine. Based on this, a label-free aptamer-based fluorescence assay for determination of Ochratoxin A(OTA) in herbal medicine was proposed with OTA aptamer as the recognition original and berberine as the fluorescent probe. For the main influencing factors, the concentrations of K^(+), Mg^(2+), berberine and aptamer had been optimized. Under the optimal experimental conditions, the change value of berberine fluorescence signal was proportional to the concentration of OTA in the range of 5-200 nmol/L, with the detection limit of 5 nmol/L. The proposed method could quantify OTA using only one unlabeled DNA strand, thus avoiding the complex structure design of aptamer. Besides, the assay exerts remarkable specificity for identifying OTA, which had been successfully applied to the determination of OTA in Platycodon grandiflorum, with a recovery of 86.3%-105.6%.

关 键 词：赭曲霉素A 核酸适配体 小檗碱 荧光 无标记 

分 类 号：O657.31[理学—分析化学] X832[理学—化学]