与草甘膦特异结合的核酸适配体筛选及应用

Screening and Application of Nucleic Acid Aptamers Specifically Binding to Glyphosate

通过体外指数富集配体系统进化(SELEX)技术,筛选靶向草甘膦核酸适配体A08。使用酶联寡核苷酸测定法(ELONA)和斑点印迹确认草甘膦核酸适配体A08与草甘膦的特异性,未观察到非特异性。基于ELONA平台,草甘膦检测限为4 ng/μL。圆二色谱(CD)实验表明,草甘膦核酸适配体A08形成茎环和分子内G-四链体,可以稳定存在于结合的磷酸盐缓冲溶液中。此外,亲和力实验显示草甘膦与核酸适配体之间具有强的结合力,解离常数(K d)为38.38±9.094 nmol/L。基于草甘膦核酸适配体A08的ELONA法测定的准确性在真正的草甘膦样品中得到证实。获得的草甘膦核酸适配体A08为制备检测草甘膦试剂盒奠定了坚实的基础。

Nucleic acid aptamers are molecules that replace the function of antibodies and can be used to develop rapid detection kits.The biggest difficulty is the selection of novel and highly specific nucleic acid aptamers.In this study,artificial nucleic acid aptamers targeting glyphosate were screened using systematic evolution of ligands by expotential enrichment(SELEX)to develop analytical tools for glyphosate detection.The specificity of aptamer A08 for glyphosate was confirmed using an enzyme-linked oligonucleotide assay(ELONA)and Dot Blot,and no nonspecificity was observed.Based on the ELONA platform,the minimum detectable concentration of glyphosate was 4 ng/μL.Circular dichroism(CD)experiments showed that aptamer A08 formed a stem loop and an intramolecular G-quadruplex,and it was stably present in binding phosphate buffer.In addition,the affinity test showed strong binding between glyphosate and the nucleic acid aptamer with a dissociation constant(K d)of 38.38±9.094 nmol/L.The accuracy of the ELONA assay based on the nucleic acid aptamer A08 was confirmed in real glyphosate samples.The obtained glyphosate nucleic acid aptamer A08 laid a solid foundation for the preparation of the glyphosate test kit.