核酸负载纳米金的改良ELISA高灵敏检测平台的构建  

Construction of modified ELISA high sensitivity detection platform by nucleic acid loaded nanogold

作　　者：程易 马粤婷 吴日红 徐瑜[1] 杨舒凌 王永霞[1] CHENG Yi;MA Yueting;WU Rihong;XU Yu;YANG Shuling;WANG Yongxia(Key Laboratory of Tropical Disease of Ministry of Education,Hainan Medical University,Haikou,Hainan 571109,China)

机构地区：[1]海南医学院教育部热带病重点实验室,海南海口571109

出　　处：《检验医学与临床》2022年第11期1441-1445,共5页Laboratory Medicine and Clinic

基　　金：国家自然科学基金项目(81560006);海南省自然科学基金高层次人才项目(820RC65);大学生创新创业项目(X201911810004)。

摘　　要：目的构建基于核酸负载纳米金(AuNPs)的酶联免疫吸附试验(ELISA)高灵敏检测平台。方法将生物素化DNA通过金-硫键结合在AuNPs上,制备纳米金生物素化DNA(AuNPs@DNA-B)信号探针,在ELISA检测目标分子的基础上,使信号探针通过链霉亲和素结合于夹心复合物中的生物素化抗体上,发挥信号放大作用,实现对目标分子的高灵敏检测。该研究选用血清水平极低的抗体免疫球蛋白(Ig)E作为目标分子,验证该检测平台的可行性。结果通过紫外光谱扫描、透射电镜观察,发现制备的AuNPs颗粒大小均匀、分散性好。在最优条件下,该方法对选定的目标分子IgE的最低检测限为0.012 ng/mL;批内变异系数<2.0%,批间变异系数<6.1%;该方法的回收率为(99.956±0.168)%~(104.733±3.376)%,传统ELISA为(99.100±0.529)%~(100.044±0.276)%,差异无统计学意义(P>0.05)。其检测灵敏度与化学发光法相近。结论该研究成功构建了基于AuNPs@DNA-B的信号放大ELISA高灵敏检测平台。Objective To construct a high sensitivity detection platform of ELISA based on the nucleic acid loaded nanogold(AuNPs).Methods The biotinylated DNA(DNA-B)was binded to AuNPs via the gold-sulfur bond.The nanogold biotinylated DNA signal probe(AuNPs@DNA-B)was prepared.On the basis of ELISA detecting the target molecule,the signal probe was combined to the biotinylated antibody in sandwich compound via streptavidin for playing the signal amplification effect and realizing the high sensitive detection on the target molecule.This study selected IgE with serum extreme low level as the target molecule to verify the feasibility of this detection platform.Results The prepared AuNPs particles were uniform with good dispersibility by the ultraviolet spectral scanning,transmission electron microscopy observation.On the optimal condition,the lowest detection limit for the selected target molecule IgE was 0.012 ng/mL.The coefficient of variation of intra-assay and inter-assay was<2.0% and<6.1%respectively.The recovery rate of this method was(99.956±0.168)%—(104.733±3.376)%,which of the traditional ELISA was(99.100±0.529)%—(100.044±0.276)%,and the differences had no statistical significance(P>0.05).Its detection sensitivity was similar to that of chemiluminescence assay.Conclusion This study successfully constructs the high sensitivity ELISA detection platform for the signal amplification based on AuNPs@DNA-B.

关 键 词：纳米金 纳米金生物素化DNA 酶联免疫吸附试验 免疫球蛋白E 

分 类 号：R392-33[医药卫生—免疫学]