基于新型核酸适配体传感器的赭曲霉毒素A检测研究

摘要

赭曲霉毒素A(OTA)是由曲霉菌属和青霉菌属等丝状真菌产生的次级代谢产物,分布广泛,已经成为普遍的食源性污染物.由于它具有肝毒性、肾毒性及致癌致畸等作用,对人们的健康构成了巨大的威胁,所以找到一种OTA的高灵敏快速检测方法对保证食品安全有至关重要的意义.电化学法具有设备简单、易携带、成本低、响应快等优点,结合核酸适配体(Apt)亲和力强、稳定性高、易于官能团修饰等优势,利用羧基化多孔碳(cPC)比表面积大等特性,以硫堇(Th)为电化学探针,制备了基于Apt和羧基化多孔碳-纳米金复合材料(cPC-AuNPs)的Th/cPC-AuNPs-cDNA/Apt/AuE传感器,用于OTA的检测研究.不同电极的交流阻抗结果表明,Apt成功固定在AuE上,Apt与cDNA成功结合.对试验参数进行优化,得到Apt的最佳孵育时间为1.5 h、Apt的最佳浓度为5 μmol/L、Apt与cDNA的最佳杂交时间为2.0 h、cDNA的最优量为9μL.在最优条件下,建立标准曲线,在10-6 ～ 10-2 μg/mL范围内,具有良好的线性关系,检出限为10-6 μg/mL.该方法操作简便、经济快速,适用于现场检测.%Ochratoxin A (OTA) is secondary metabolite produced by filamentous fungi of Aspergillus and Penicillium,and it is widely distributed and has become a common food borne contaminant.Ochratoxin A (OTA) has liver toxicity,nephrotoxicity and carcinogenic teratogenic effects,and has huge threat to the human health.Therefore,it is very important to find a sensitive and rapid OTA detection method for ensuring food safety.In this paper,a Th/cPC-AuNPs-cDNA/Apt/AuE sensor for rapid detection of OTA was prepared by using thionine (Th) as electrochemical probe based on aptamer and cPC-AuNPs;and the Th/cPC-AuNPs-cDNA/Apt/AuE sensor possessed the advantages of electrochemical method such as simple equipment,portability,low cost,and quick response,the superiorities of aptamer such as high affinity,high stability and easy modification of functional groups,as well as the characteristics of carboxylated porous carbons such as large specific area.The results of electrochemical impedance spectroscopy showed that the aptamer was successfully immobilized on AuE and combined with cDNA.The experimental parameters were optimized as follows:the optimum incubation time of aptamer was 1.5 h,the optimum concentration of aptamer was 5 μmoL/L,the optimum hybridization time between aptamer and cDNA was 2 h,and the optimum amount of cDNA was 9 μL.Under the optimal conditions,the standard curve was established,which had good linear relationship within 10-6 μg/mL to 10-2 μg/mL,and the detection limit of OTA was l0-6 μg/mL.The mehtod is easy to operate,economic,fast,and is suitable for filed detection.