Ultra-Sensitive Detection of the SARS-CoV-2 Nucleocapsid Protein via a Clustered Regularly Interspaced Short Palindromic Repeat/Cas12a-Mediated Immunoassay

文献类型： 外文期刊

第一作者： Yin, Wen

作者： Yin, Wen;Li, Leyao;Ma, Lixin;Yang, Yang;Yang, Yuxin;Dai, Junbiao;Mao, Guobin;Ma, Yingxin;Dai, Junbiao;Liang, Ruijing

作者机构：

关键词： ultrasensitive detection; immunoassay; SARS-CoV-2N protein; CRISPR/Cas12a; RCA

期刊名称：ACS SENSORS （ 影响因子：8.9； 五年影响因子：9.0 ）

ISSN： 2379-3694

年卷期： 2024 年

页码：

收录情况： SCI

摘要： Tracking trace protein analytes in precision diagnostics is an ongoing challenge. Here, we developed an ultrasensitive detection method for the detection of SARS-CoV-2 nucleocapsid (N) protein by combining enzyme-linked immunosorbent assay (ELISA) with the clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas) system. First, the SARS-CoV-2 N protein bound by the capture antibody adsorbed on the well plate was sequentially coupled with the primary antibody, biotinylated secondary antibody, and streptavidin (SA), followed by biotin primer binding to SA. Subsequently, rolling circle amplification was initiated to generate ssDNA strands, which were targeted by CRISPR/Cas12a to cleave the FAM-ssDNA-BHQ1 probe in trans to generate fluorescence signals. We observed a linear relationship between fluorescence intensity and the logarithm of N protein concentration ranging from 3 fg/mL to 3 x 10(7) fg/mL. The limit of detection (LOD) was 1 fg/mL, with approximately nine molecules in 1 mu L of the sample. This detection sensitivity was 4 orders magnitude higher than that of commercially available ELISA kits (LOD: 5.7 x 10(4) fg/mL). This method was highly specific and sensitive and could accurately detect SARS-CoV-2 pseudovirus and clinical samples, providing a new approach for ultrasensitive immunoassay of protein biomarkers.

分类号：

  • 相关文献
作者其他论文 更多>>

微信小程序