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

Yin, Wen1; Li, Leyao1; Yang, Yang2; Yang, Yuxin3; Liang, Ruijing5; Ma, Lixin1; Dai, Junbiao3,4; Mao, Guobin3; Ma, Yingxin3

2024-05-08

10.1021/acssensors.4c00432

ACS SENSORS   影响因子和分区

2379-3694

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.

ultrasensitive detection immunoassay SARS-CoV-2N protein CRISPR/Cas12a RCA

SCI ; EI

英语

其他

National Key Research and Development Program of China[2021YFA0910900] ; National Natural Science Foundation of China["32222044","22104147","32000055"] ; Shenzhen Municipal Science and Technology Innovation Council[RCYX20210609103823046] ; Youth Innovation Promotion Association CAS[2021359] ; Guangdong Provincial Key Laboratory of Synthetic Genomics[2023B1212060054] ; Shenzhen Key Laboratory of Synthetic Genomics[ZDSYS201802061806209] ; Shenzhen Science and Technology Program[KQTD20180413181837372]

Chemistry ; Science & Technology - Other Topics

Chemistry, Multidisciplinary ; Chemistry, Analytical ; Nanoscience & Nanotechnology

WOS:001225131600001

AMER CHEMICAL SOC

Web of Science

1.Hubei Univ, Sch Life Sci, State Key Lab Biocatalysis & Enzyme Engn, Hubei Key Lab Ind Biotechnol, Wuhan 430062, Peoples R China
2.Southern Univ Sci & Technol, Shenzhen Peoples Hosp 3, Natl Clin Res Ctr Infect Dis, Shenzhen Key Lab Pathogen & Immun,State Key Discip, Shenzhen 518112, Peoples R China
3.Chinese Acad Sci, Shenzhen Inst Synthet Biol, Shenzhen Inst Adv Technol, Shenzhen Key Lab Synthet Genom,Guangdong Prov Key, Shenzhen 518055, Peoples R China
4.Chinese Acad Agr Sci, Agr Genom Inst Shenzhen, Shenzhen Branch, Guangdong Lab Lingnan Modern Agr,Key Lab Synthet B, Shenzhen 518120, Peoples R China
5.Chinese Acad Sci, Guangdong Key Lab Nanomed, CAS HK Joint Lab Biomat, Shenzhen Inst Adv Technol,Inst Biomed & Biotechnol, Shenzhen 518055, Peoples R China

Yin, Wen,Li, Leyao,Yang, Yang,et al. Ultra-Sensitive Detection of the SARS-CoV-2 Nucleocapsid Protein via a Clustered Regularly Interspaced Short Palindromic Repeat/Cas12a-Mediated Immunoassay[J]. ACS SENSORS,2024,9(6).

Yin, Wen.,Li, Leyao.,Yang, Yang.,Yang, Yuxin.,Liang, Ruijing.,...&Ma, Yingxin.(2024).Ultra-Sensitive Detection of the SARS-CoV-2 Nucleocapsid Protein via a Clustered Regularly Interspaced Short Palindromic Repeat/Cas12a-Mediated Immunoassay.ACS SENSORS,9(6).

Yin, Wen,et al."Ultra-Sensitive Detection of the SARS-CoV-2 Nucleocapsid Protein via a Clustered Regularly Interspaced Short Palindromic Repeat/Cas12a-Mediated Immunoassay".ACS SENSORS 9.6(2024).