Ratiometric fluorescence immunoassay of SARS-CoV-2 nucleocapsid protein via Si-FITC nanoprobe-based inner filter effect

Guobin Mao; Yang Yang; Shijie Cao; Silu Ye; Yifang Li; Wei Zhao; Hongwei An; Yingxia Liu; Junbiao Dai; Yingxin Ma

doi:10.1007/s12274-022-4740-5

Ratiometric fluorescence immunoassay of SARS-CoV-2 nucleocapsid protein via Si-FITC nanoprobe-based inner filter effect

Nano Res. 2023;16(4):5383-5390. doi: 10.1007/s12274-022-4740-5. Epub 2022 Aug 17.

Authors

Guobin Mao^#¹, Yang Yang^#², Shijie Cao^#^{1

3}, Silu Ye¹, Yifang Li¹, Wei Zhao¹, Hongwei An³, Yingxia Liu², Junbiao Dai¹, Yingxin Ma¹

Affiliations

¹ CAS Key Laboratory of Quantitative Engineering Biology, Guangdong Provincial Key Laboratory of Synthetic Genomics and Shenzhen Key Laboratory of Synthetic Genomics, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055 China.
² Shenzhen Key Laboratory of Pathogen and Immunity, National Clinical Research Center for infectious disease, State Key Discipline of Infectious Disease, Shenzhen Third People's Hospital, Second Hospital Affiliated to Southern University of Science and Technology, Shenzhen, 518112 China.
³ Guangxi University of Chinese Medicine, Nanning, 530001 China.

^# Contributed equally.

Abstract

The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has necessitated rapid, easy-to-use, and accurate diagnostic methods to monitor the virus infection. Herein, a ratiometric fluorescence enzyme-linked immunosorbent assay (ELISA) was developed using Si-fluorescein isothiocyanate nanoparticles (FITC NPs) for detecting SARS-CoV-2 nucleocapsid (N) protein. Si-FITC NPs were prepared by a one-pot hydrothermal method using 3-aminopropyl triethoxysilane (APTES)-FITC as the Si source. This method did not need post-modification and avoided the reduction in quantum yield and stability. The p-nitrophenyl (pNP) produced by the alkaline phosphatase (ALP)-mediated hydrolysis of p-nitrophenyl phosphate (pNPP) could quench Si fluorescence in Si-FITC NPs via the inner filter effect. In ELISA, an immunocomplex was formed by the recognition of capture antibody/N protein/reporter antibody. ALP-linked secondary antibody bound to the reporter antibody and induced pNPP hydrolysis to specifically quench Si fluorescence in Si-FITC NPs. The change in fluorescence intensity ratio could be used for detecting N protein, with a wide linearity range (0.01-10.0 and 50-300 ng/mL) and low detection limit (0.002 ng/mL). The concentration of spiked SARS-CoV-2 N protein could be determined accurately in human serum. Moreover, this proposed method can accurately distinguish coronavirus disease 2019 (COVID-19) and non-COVID-19 patient samples. Therefore, this simple, sensitive, and accurate method can be applied for the early diagnosis of SARS-CoV-2 virus infection.

Electronic supplementary material: Supplementary material (characterization of Si-FITC NPs (FTIR spectrum, XRD spectra, and synchronous fluorescence spectra); condition optimization of ALP response (fluorescence intensity ratio change); mechanism investigation of ALP response (fluorescence lifetime decay curves and UV-vis absorption spectra); detection of N protein using commercial ELISA Kit; analytical performance of assays for ALP detection or SARS-CoV-2 N protein detection; and determination results of SARS-CoV-2 N protein in human serum) is available in the online version of this article at 10.1007/s12274-022-4740-5.

Keywords: Si-fluorescein isothiocyanate (FITC) nanoparticles; enzyme-linked immunosorbent assay (ELISA); inner filter effect; ratiometric fluorescent probe; severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).