基于适配体/小檗碱荧光体系的赭曲霉素A检测方法研究及在中药中的应用

Ochratoxin A (OTA) is a mycotoxin which exists widely in traditional Chinese medicine and affects seriously the safety of traditional Chinese medicine. At present, there are two methods to detect OTA in traditional Chinese medicine, one uses the large instruments such as chromatography, which requires complex sample pretreatment, the other is antibody-based immunoassay, but the antibody stability is poor. As a new type of recognition element, apatmers can be tightly bound to the target molecules in a complex matrix and suffer less interference. Aptamers have been widely studied in many fields because of their advantage of good stability, strong specificity, high sensitivity and so on..Based on our good aptamer detection technology and the characteristic that the fluorescent signal being obviously enhanced after the berberine combining with the aptamer with the G - quadruplex structure, this project intends to develop several new label-free methods for Ochratoxin detection using aptamer/berberiner fluorescence systems with aptamers as identification elements and berberine as fluorescence probe. The project intends to detect OTA in the malt samples without complex pretreatment, and the accuracy of the method will be confirmed by liquid chromatography-mass spectrometry (LC-MS). This study will be applied to the detection of OTA in a variety of traditional Chinese medicines in order to expand the scope of application of aptamers in traditional Chinese medicine, and provide a reference for OTA detection in traditional Chinese medicine, ensure the safety of clinical medication of traditional Chinese medicine.

赭曲霉素A（OTA）是一种真菌毒素，广泛存在于中药中，严重影响中药用药安全。目前，中药中OTA的检测主要有两种方法，一种是使用大型仪器如色谱法，这需要复杂的样品前处理，另一种是基于抗体的免疫分析法，但抗体稳定性差。适配体作为一种新型识别元件，能与复杂基质中微量的靶分子紧密结合而受到较少干扰，具有稳定性好，特异性强等优势，在许多领域被广泛研究。.本项目以我们良好的适配体检测技术为基础，拟以适配体为识别元件，小檗碱为荧光探针，利用小檗碱与G-四链体结构的适配体作用后荧光信号显著增强的特性，构建几种基于适配体/小檗碱免标记荧光体系检测OTA的新方法，项目拟以中药麦芽为例，不经过复杂的样品前处理，实现中药实际样品中OTA的检测。并用液相-质谱联用法(LC-MS)确证方法的准确性。此研究将应用到多种中药中，以期拓展适配体在中药的应用范围，为更简便快捷的检测OTA，保障中药临床用药安全提供参考。

我们利用适配体和赭曲霉素A特异性结合后引起荧光探针（小檗碱或荧光素FAM）的荧光强度变化，建立了三种适配体/荧光体系定量检测赭曲霉素A，包括基于小檗碱/适配体免标记荧光体系、基于G碱基猝灭的免标记适配体/荧光体系和基于磁球的单标记适配体/荧光体系。三种方法且均成功用于中药中赭曲霉素A的测定。我们也采用酶放大技术，发展了凝血酶联适配体法检测蛋白质，并用于人血清中，为进一步构建高灵敏度适配体/荧光体系检测赭曲霉素A提供了新方法。我们以荧光光谱法和荧光偏振法等考察了DNA对镍卟啉荧光强度和荧光偏振值的影响，为拓展新型荧光探针用于适配体/荧光体系奠定了理论基础。我们建立的方法具有良好的选择性和高的灵敏度，且具有一定的普遍性，为中药质量鉴定提供了新思路。