基于孔雀石绿/核酸适配体的免标记荧光法检测中药中的赭曲霉素A  

A fluorescence method based on malachite green/aptamer for detection of ochratoxin A in traditional Chinese medicines

作　　者：杨圆圆 邓峥嵘 高仕超 郭丽敏 YANG Yuan-yuan;DENG Zheng-rong;GAO Shi-chao;GUO Li-min(Shanxi University of Chinese Medicine,Jinzhong 030619,China)

机构地区：[1]山西中医药大学,山西晋中030619

出　　处：《中国中药杂志》2024年第7期1818-1825,共8页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(81803725);山西省中医药科研课题(2022ZYYC107);山西中医药大学博士启动金基金项目(2023BK42);山西中医药大学科技创新能力培育计划项目(2022PY-TH-08)。

摘　　要：该文报道了一种基于孔雀石绿/核酸适配体的免标记荧光法检测中药中的赭曲霉素A(OTA)。孔雀石绿本身荧光信号微弱,与赭曲霉素A的核酸适配体作用后,G-四链体结构的核酸适配体为孔雀石绿提供一个保护的微环境,使其荧光信号显著增强。再加入OTA之后,核酸适配体会优先与OTA结合,从而使孔雀石绿游离于核酸适配体之外,其荧光信号随之减弱。基于此,该文以OTA的核酸适配体为识别元件,孔雀石绿为荧光探针,建立了一种基于核酸适配体的荧光法检测中药中的OTA。并对金属离子、核酸适配体和孔雀石绿的浓度等影响实验结果的主要因素进行了进一步的优化。在最佳实验条件下对OTA进行了检测,实验发现,随着OTA浓度的增大,荧光信号逐渐减弱,在20~1000 nmol·L^(-1),OTA浓度与荧光信号比值ΔF/F呈线性相关(ΔF=F_(0)-F,F_(0)为核酸适配体/孔雀石绿的荧光信号,F为OTA/核酸适配体/孔雀石绿的荧光信号),检出限为7.1 nmol·L^(-1),R^(2)=0.995。为考察该方法的特异性,该实验选择了3种与OTA结构相似的物质以及2种可能和OTA共存的真菌毒素进行交叉反应率实验和特异性实验。交叉反应率实验表明这些干扰物不会引起检测体系明显的荧光信号变化,而特异性实验显示了当干扰物与OTA混合后,混合物产生的荧光信号与OTA的荧光信号接近,说明即使与干扰物共存,OTA的检测也不会受到影响,说明方法具有良好的特异性;方法也展现了良好的重复性和稳定性。仅对中药进行简单的离心和稀释,方法即可用于中药葛根、苦参、香加皮中OTA的检测,回收率在91.50%~121.3%。该方法不需要复杂的中药预处理,操作简单,检测成本低,检测时间短,而且方法将核酸适配体应用于中药质量鉴定中,进一步拓展了核酸适配体的应用范围。A label-free fluorescence method based on malachite green/aptamer was developed for the detection of ochratoxin A(OTA)in traditional Chinese medicines.Malachite green itself exhibits weak fluorescence.Upon interaction with the aptamer specific to OTA,the G-quadruplex structure of the aptamer provides a protective microenvironment for malachite green,which significantly enhances its fluorescence signal.After OTA is added,preferential binding occurs between the aptamer and OTA,and malachite green will be released from the aptamer,which weakens the fluorescence signal.According to this principle,this paper established a fluorescence method with the aptamer of OTA as the recognition element and malachite green as the fluorescent probe for the detection of OTA in traditional Chinese medicines.The key experimental factors such as the concentrations of metal ions,aptamer,and malachite green were optimized to improve the performance of the method.OTA was detected under the optimal experimental conditions,and the results showed that with the increase in OTA concentration,the fluorescence signal gradually weakened.Within the range of 20-1000 nmol·L^(-1),the OTA concentration was linearly correlated with the fluorescence signal ratioΔF/F(ΔF=F_(0)-F,where F_(0)is the fluorescence signal of aptamer/malachite green,and F is the fluorescence signal of OTA/aptamer/malachite green),with R^(2)of 0.995.The limit of detection of the established method was 7.1 nmol·L^(-1).Furthermore,three substances structurally similar to OTA and two mycotoxins that may coexist with OTA were selected for experiments,which aimed to examine the crossreactivity and specificity of the established method.The cross-reactivity experiments demonstrated that the interferers did not significantly affect the fluorescence signal of the detection system.The specificity experiments revealed that when mycotoxins were mixed with OTA,the fluorescence signal generated by the mixture closely resembled that of OTA itself.The results indicated that even in the pres

关 键 词：核酸适配体 孔雀石绿 赭曲霉素A 荧光 免标记 

分 类 号：R284.1[医药卫生—中药学] O657.3[医药卫生—中医学]