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

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.