基于SERS免疫层析技术的人腺病毒抗原检测方法建立

Establishment of detection method for human adenovirus antigen via surface-enhanced Raman scattering immunochromatographic assay

目的基于Au@Ag NPs的SERS侧流免疫层析(LFIA)建立一种快速、高灵敏定量检测人腺病毒(HAdV)的方法。方法首先合成出双层拉曼分子5, 5’-二硫代双(2-硝基苯甲酸) (DTNB)修饰的Au@Ag NPs, 再结合HAdV特异性抗体得到SERS探针, 基于抗原-抗体的特异性反应在免疫层析条检测线处形成"三明治"免疫复合结构, 通过收集并分析检测线上的SERS信号, 可以实现对HAdV抗原的快速检测。其次, 分别对该检测方法的灵敏度、重复性和特异性进行评价。结果基于双层DTNB修饰的Au@Ag NPs SERS-LFIA可在15 min内对HAdV实现定性/定量检测, 其可视化检测结果为10 ng/ml, 最低检出限达0.1 ng/ml, 定量范围为0.1 ng/ml~1 000 ng/ml。此SERS免疫层析方法对HAdV的检测具有良好的重复性和特异性, 与登革热病毒、黄热病毒、西尼罗河病毒、寨卡病毒和埃博拉病毒五种新突发传染性病毒均无交叉反应。结论该研究提出的基于Au/DTNB@Ag/DTNB NPs的SERS-LFIA可以实现对HAdV的快速和高灵敏检测, 在HAdV的现场即时检测领域具有巨大潜力。

Objective To establish a rapid and ultrasensitive detection of human adenovirus(HAdV)antigen via Au@Ag NPs-based SERS lateral flow immunoassay(LFIA).Methods Firstly,the Au@Ag NPs modified with dual-layer 5,5’-dithiobis-(2-nitrobenzoic acid)(DTNB)was synthesized,and conjugated with specific anti-HAdV antibodies to prepare SERS tags.Next,the SERS tags,HAdV and specific detecting antibodies of anti-HAdV on the test lines(T lines)of strips formed the complex structure of sandwich based on the reaction of antigen and antibody,and the rapid detection of HAdV can be achieved by collecting and analyzing the SERS signals on the T lines.Next,the sensitivity,reproductivity and specificity of the method were evaluated,respectively.Results The proposed SERS-LFIA via dual-layer DTNB-modified Au@Ag NPs can achieve the qualitative and quantitative detection of HAdV within 15 min.The visual result was 10 ng/ml,and limit of detection(LOD)was 0.1 ng/ml with the quantitative range from 0.1 ng/mL to 1000 ng/ml.Furthermore,this SERS-LFIA displayed great reproducibility and specificity,and there was no cross-reaction with other five emerging infectious virues including dengue virus,yellow fever virus,west nile virus,zika virus and ebola virus.Conclusions In this study,the proposed SERS-LFIA based on Au/DTNB@Ag/DTNB NPs can accomplish rapid and ultrasensitive detection of HAdV,revealing the great potential in the field of point of care testing.