Abstract：
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 20-1 000 nmol·L~(-1), the OTA concentration was linearly correlated with the fluorescence signal ratio ΔF/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 cross-reactivity 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 presence of interferents, the established method remained unaffected and demonstrated excellent specificity. Additionally, this method exhibited remarkable reproducibility and stability. In the case of simple centrifugation and dilution of traditional Chinese medicine samples(Puerariae Lobatae Radix, Sophorae Flavescentis Radix, and Periplocae Cortex), the OTA detection method was applicable, with recovery rates ranging from 91.5% to 121.3%. Notably, this approach does not need complex pretreatment of traditional Chinese medicines while offering simple operation, low detection costs, and short detection time. Furthermore, by incorporating aptamers into the quality evaluation of traditional Chinese medicines, this method expands the application scope of aptamers.
摘要：
建立了一种基于孔雀石绿/适配体的无标记荧光法检测中药中曲霉毒素A(OTA)的方法。孔雀石绿本身表现出微弱的荧光。在与OTA特异性适体相互作用时，适体的G-四链体结构为孔雀石绿提供了保护性的微环境，显着增强其荧光信号。添加OTA后,优先结合发生在适体和OTA之间，孔雀石绿将从适体中释放出来，这削弱了荧光信号。根据这一原则,本文建立了以OTA的适体为识别元素,孔雀石绿为荧光探针的荧光检测方法。金属离子浓度等关键实验因素,适体,和孔雀石绿进行了优化,以提高方法的性能。在最佳实验条件下检测到OTA，结果表明,随着OTA浓度的增加,荧光信号逐渐减弱。在20-1000nmol·L~(-1)范围内，OTA浓度与荧光信号比ΔF/F呈线性关系(ΔF=F_0-F,其中F_0是适体/孔雀石绿的荧光信号，F为OTA/适体/孔雀石绿的荧光信号),R~2为0.995。方法的检出限为7.1nmol·L~(-1)。此外，选择了三种结构上与OTA相似的物质和两种可能与OTA共存的霉菌毒素进行实验，旨在检查所建立方法的交叉反应性和特异性。交叉反应性实验表明，干扰源不会显着影响检测系统的荧光信号。特异性实验表明，当真菌毒素与OTA混合时，由混合物产生的荧光信号非常类似于OTA本身。结果表明，即使存在干扰物，所建立的方法未受影响,并表现出优异的特异性.此外,该方法具有显著的重现性和稳定性。在简单离心和稀释中药样品（葛根，苦参,和骨膜皮层），OTA检测方法适用，回收率从91.5%到121.3%。值得注意的是,这种方法不需要复杂的中药预处理，同时提供简单的操作，低检测成本，和短的检测时间。此外，通过将适体纳入中药质量评价，该方法扩大了适体的应用范围。
