基于氧化石墨烯的磺胺嘧啶核酸适配体的筛选与鉴定

Screening and Identification of Sulfadiazine Aptamer Based on Graphene Oxide

【目的】利用GO-SELEX方法对磺胺嘧啶(Sulfadiazine)核酸适配体进行筛选与鉴定。【方法】基于氧化石墨烯(Graphene oxide)与ssDNA的π-π堆积作用,将氧化石墨烯作为结合ssDNA与分离的介质,以磺胺嘧啶为靶标分子,利用指数富集的配体系统进化技术(SELEX)和磁分离技术建立了基于氧化石墨烯的核酸适配体筛选方法 GO-SELEX,对筛选产物测序所得的核酸序列进行了同源性分析和二级结构预测,亲和力和特异性分析以及方法学考察。【结果】经过9轮筛选,获得了对磺胺嘧啶具有较高亲和力(Kd=411nmol·L^(-1))的核酸适配体Ap-41,且Ap-41对磺胺嘧啶的特异性良好;磺胺嘧啶质量浓度在1.0~40μg·mL^(-1)范围内与荧光强度的线性关系良好,检出限为0.029μg·mL^(-1),水样中加标回收率范围为95.0%~104.0%,相对标准偏差不超过6.1%。【结论】基于核酸适配体Ap-41的检测方法灵敏度较高,可用于后续传感器的构建及实际样品中磺胺嘧啶的检测。

[Purposes]It aims to screen and identify the aptamer of sulfadiazine by GO-SELEX method.[Methods]Based on theπ-πstacking of graphene oxide(GO)and ssDNA,using GO as the medium to combine ssDNA and separation.Using sulfadiazine as the target molecule,a new method of aptamer selection based on graphene oxide(GO-SELEX)was established by using the systematic evolution of ligands by exponential enrichment(SELEX)and magnetic separation technology.Homology analysis,secondary structure prediction,affinity and specificity analysis as well as methodology investigation were carried out for the nucleic acid sequences obtained from the sequencing of the screened products.[Findings]Ap-41 with high affinity for sulfadiazine(Kd=411 nmol·L^(-1))was obtained through nine rounds of screening,and the specificity of Ap-41 for sulfadiazine was good.The linear relationship between sulfadiazine concentration in the range of 1.0~40μg·mL^(-1) and fluorescence intensity was good,the detection limit was 0.029μg·mL^(-1),the recovery of sulfadiazine in water samples was 95.0%~104.0%,and the relative standard deviation was not more than 6.1%.[Conclusions]The detection method based on aptamer Ap-41 has high sensitivity and can be used for the construction of subsequent sensors and the detection of sulfadiazine in actual samples.