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Nano Research 2023, 16(4): 5383-5390 https://doi.org/10.1007/s12274-022-4740-5
Research Article | Issue | Published: 17 August 2022

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

Show Author's Information Guobin Mao1,§,  Yang Yang3,§,  Shijie Cao1,2,§,  Silu Ye1,  Yifang Li1,  Wei Zhao1,  Hongwei An2,  Yingxia Liu3( ),  Junbiao Dai1,  Yingxin Ma1( )
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
Guangxi University of Chinese Medicine, Nanning 530001, China
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

§ Guobin Mao, Yang Yang, and Shijie Cao contributed equally to this work.

Keywords:
severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Si-fluorescein isothiocyanate (FITC) nanoparticles, ratiometric fluorescent probe, inner filter effect, enzyme-linked immunosorbent assay (ELISA)
Topics:
Fluorescence spectroscopy
Cite this article:
Mao G, Yang Y, Cao S, et al. Ratiometric fluorescence immunoassay of SARS-CoV-2 nucleocapsid protein via Si-FITC nanoprobe-based inner filter effect. Nano Research, 2023, 16(4): 5383-5390. https://doi.org/10.1007/s12274-022-4740-5
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Abstract Full text Electronic supplementary material About this article

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.


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Abstract
Full text
Outline
Electronic supplementary material
About this article

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

Show Author's information Guobin Mao1,§, Yang Yang3,§, Shijie Cao1,2,§, Silu Ye1, Yifang Li1, Wei Zhao1, Hongwei An2, Yingxia Liu3( ), Junbiao Dai1, Yingxin Ma1( )
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
Guangxi University of Chinese Medicine, Nanning 530001, China
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

§ Guobin Mao, Yang Yang, and Shijie Cao contributed equally to this work.

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.

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

References(34)

[1]

Ma, Y. X.; Mao, G. B.; Huang, W. R.; Wu, G. Q.; Yin, W.; Ji, X. H.; Deng, Z. S.; Cai, Z. M.; Zhang, X. E.; He, Z. K. et al. Quantum dot nanobeacons for single RNA labeling and imaging. J. Am. Chem. Soc. 2019, 141, 13454–13458.
DOI Google Scholar
[2]

Devi, P.; Saini, S.; Kim, K. H. The advanced role of carbon quantum dots in nanomedical applications. Biosens. Bioelectron. 2019, 141, 111158.
DOI Google Scholar
[3]

Zhong, Y. L.; Peng, F.; Bao, F.; Wang, S. Y.; Ji, X. Y.; Yang, L.; Su, Y. Y.; Lee, S. T.; He, Y. Large-scale aqueous synthesis of fluorescent and biocompatible silicon nanoparticles and their use as highly photostable biological probes. J. Am. Chem. Soc. 2013, 135, 8350–8356.
DOI Google Scholar
[4]

So, W. Y.; Li, Q.; Legaspi, C. M.; Redler, B.; Koe, K. M.; Jin, R. C.; Peteanu, L. A. Mechanism of ligand-controlled emission in silicon nanoparticles. ACS Nano 2018, 12, 7232–7238.
DOI Google Scholar
[5]

Han, Y. X.; Chen, Y. L.; Liu, J. J.; Niu, X. Y.; Ma, Y. X.; Ma, S. D.; Chen, X. G. Room-temperature synthesis of yellow-emitting fluorescent silicon nanoparticles for sensitive and selective determination of crystal violet in fish tissues. Sens. Actuators B:Chem. 2018, 263, 508–516.
DOI Google Scholar
[6]

Zhou, Y. F.; Zhang, Y.; Zhong, Y. L.; Fu, R.; Wu, S. C.; Wang, Q.; Wang, H. Y.; Su, Y. Y.; Zhang, H. M.; He, Y. The in vivo targeted molecular imaging of fluorescent silicon nanoparticles in Caenorhabditis elegans. Nano Res. 2018, 11, 2336–2346.
DOI Google Scholar
[7]

Han, J. F.; Zhang, L.; Cui, M. Y.; Su, Y. Y.; He, Y. Rapid and accurate detection of lymph node metastases enabled through fluorescent silicon nanoparticles-based exosome probes. Anal. Chem. 2021, 93, 10122–10131.
DOI Google Scholar
[8]

Jiao, Y.; Gao, Y. F.; Meng, Y. T.; Lu, W. J.; Liu, Y.; Han, H.; Shuang, S. M.; Li, L.; Dong, C. One-step synthesis of label-free ratiometric fluorescence carbon dots for the detection of silver ions and glutathione and cellular imaging applications. ACS Appl. Mater. Interfaces 2019, 11, 16822–16829.
DOI Google Scholar
[9]

Park, S. H.; Kwon, N.; Lee, J. H.; Yoon, J.; Shin, I. Synthetic ratiometric fluorescent probes for detection of ions. Chem. Soc. Rev. 2020, 49, 143–179.
DOI Google Scholar
[10]

Yue, Y. K.; Huo, F. J.; Ning, P.; Zhang, Y. B.; Chao, J. B.; Meng, X. M.; Yin, C. X. Dual-site fluorescent probe for visualizing the metabolism of Cys in living cells. J. Am. Chem. Soc. 2017, 139, 3181–3185.
DOI Google Scholar
[11]

Lan, M. H.; Zhang, J. F.; Chui, Y. S.; Wang, P. F.; Chen, X. F.; Lee, C. S.; Kwong, H. L.; Zhang, W. J. Carbon nanoparticle-based ratiometric fluorescent sensor for detecting mercury ions in aqueous media and living cells. ACS Appl. Mater. Interfaces 2014, 6, 21270–21278.
DOI Google Scholar
[12]

Zou, C. C.; Foda, M. F.; Tan, X. C.; Shao, K.; Wu, L.; Lu, Z. C.; Bahlol, H. S.; Han, H. Y. Carbon-dot and quantum-dot-coated dual-emission core-satellite silica nanoparticles for ratiometric intracellular Cu2+ imaging. Anal. Chem. 2016, 88, 7395–7403.
DOI Google Scholar
[13]

Mao, G. B.; Cai, Q.; Wang, F. B.; Luo, C. L.; Ji, X. H.; He, Z. K. One-step synthesis of Rox-DNA functionalized CdZnTeS quantum dots for the visual detection of hydrogen peroxide and blood glucose. Anal. Chem. 2017, 89, 11628–11635.
DOI Google Scholar
[14]

Ma, J. W.; Chen, Y. G.; Chen, L.; Wang, L. Y. Ternary Pd-Ni-P nanoparticle-based nonenzymatic glucose sensor with greatly enhanced sensitivity achieved through active-site engineering. Nano Res. 2017, 10, 2712–2720.
DOI Google Scholar
[15]

Chu, B. B.; Wang, H. Y.; Song, B.; Peng, F.; Su, Y. Y.; He, Y. Fluorescent and photostable silicon nanoparticles sensors for real-time and long-term intracellular pH measurement in live cells. Anal. Chem. 2016, 88, 9235–9242.
DOI Google Scholar
[16]

Yan, X.; Li, H. X.; Han, X. S.; Su, X. G. A ratiometric fluorescent quantum dots based biosensor for organophosphorus pesticides detection by inner-filter effect. Biosens. Bioelectron. 2015, 74, 277–283.
DOI Google Scholar
[17]

Li, H. Y.; Lin, H. Y.; Lv, W. X.; Gai, P. P.; Li, F. Equipment-free and visual detection of multiple biomarkers via an aggregation induced emission luminogen-based paper biosensor. Biosens. Bioelectron. 2020, 165, 112336.
DOI Google Scholar
[18]

Zhang, J. Y.; Zhou, R. H.; Tang, D. D.; Hou, X. D.; Wu, P. Optically-active nanocrystals for inner filter effect-based fluorescence sensing: Achieving better spectral overlap. TrAC Trends Anal. Chem. 2019, 110, 183–190.
DOI Google Scholar
[19]

Zhai, W. Y.; Wang, C. X.; Yu, P.; Wang, Y. X.; Mao, L. Q. Single-layer MnO2 nanosheets suppressed fluorescence of 7-hydroxycoumarin: Mechanistic study and application for sensitive sensing of ascorbic acid in vivo. Anal. Chem. 2014, 86, 12206–12213.
DOI Google Scholar
[20]

Li, X.; Liu, P.; Niu, X. H.; Ye, K.; Ni, L.; Du, D.; Pan, J. M.; Lin, Y. H. Tri-functional Fe-Zr bi-metal-organic frameworks enable high-performance phosphate ion ratiometric fluorescent detection. Nanoscale 2020, 12, 19383–19389.
DOI Google Scholar
[21]

Zhu, N.; Zhang, D. Y.; Wang, W. L.; Li, X. W.; Yang, B.; Song, J. D.; Zhao, X.; Huang, B. Y.; Shi, W. F.; Lu, R. J. et al. A novel coronavirus from patients with pneumonia in China, 2019. N. Engl. J. Med. 2020, 382, 727–733.
DOI Google Scholar
[22]

Ravi, N.; Cortade, D. L.; Ng, E.; Wang, S. X. Diagnostics for SARS-CoV-2 detection: A comprehensive review of the FDA-EUA COVID-19 testing landscape. Biosens. Bioelectron. 2020, 165, 112454.
DOI Google Scholar
[23]
Wang, Y.; Yan, T. H.; Mei, K. N.; Rao, D. P.; Wu, W. J.; Chen, Y.; Peng, Y. P.; Wang, J. Y.; Wu, S. Q.; Zhang, Q. C. Nanomechanical assay for ultrasensitive and rapid detection of SARS-CoV-2 based on peptide nucleic acid. Nano Res., in press, https://doi.org/10.1007/s12274-022-4333-3.
[24]

Jang, A. S.; Kumar, P. P. P.; Lim, D. K. Attomolar sensitive magnetic microparticles and a surface-enhanced Raman scattering-based assay for detecting SARS-CoV-2 nucleic acid targets. ACS Appl. Mater. Interfaces 2022, 14, 138–149.
DOI Google Scholar
[25]

Chen, M. R.; Cui, D. Z.; Zhao, Z. Y.; Kang, D.; Li, Z.; Albawardi, S.; Alsageer, S.; Alamri, F.; Alhazmi, A.; Amer, M. R. et al. Highly sensitive, scalable, and rapid SARS-CoV-2 biosensor based on In2O3 nanoribbon transistors and phosphatase. Nano Res. 2022, 15, 5510–5516.
DOI Google Scholar
[26]

Mao, G. B.; Zhang, Q.; Yang, Y. L.; Ji, X. H.; He, Z. K. Facile synthesis of stable CdTe/CdS QDs using dithiol as surface ligand for alkaline phosphatase detection based on inner filter effect. Anal. Chim. Acta 2019, 1047, 208–213.
DOI Google Scholar
[27]

Chen, W.; Habibul, N.; Liu, X. Y.; Sheng, G. P.; Yu, H. Q. FTIR and synchronous fluorescence heterospectral two-dimensional correlation analyses on the binding characteristics of copper onto dissolved organic matter. Environ. Sci. Technol. 2015, 49, 2052–2058.
DOI Google Scholar
[28]

Zhao, D.; Li, J.; Peng, C. Y.; Zhu, S. Y.; Sun, J.; Yang, X. R. Fluorescence immunoassay based on the alkaline phosphatase triggered in situ fluorogenic reaction of o-phenylenediamine and ascorbic acid. Anal. Chem. 2019, 91, 2978–2984.
DOI Google Scholar
[29]

Wei, Q. S.; Zhou, D. X.; Li, X. Q.; Chen, Y. W.; Bian, H. T. Structural dynamics of dimethyl sulfoxide aqueous solutions investigated by ultrafast infrared spectroscopy: Using thiocyanate anion as a local vibrational probe. J. Phys. Chem. B 2018, 122, 12131–12138.
DOI Google Scholar
[30]

Vu, H. P.; Moreau, J. W. Thiocyanate adsorption on ferrihydrite and its fate during ferrihydrite transformation to hematite and goethite. Chemosphere 2015, 119, 987–993.
DOI Google Scholar
[31]

Castro, M. C.; Gurzenda, S.; Turra, C. M.; Kim, S.; Andrasfay, T.; Goldman, N. Reduction in life expectancy in Brazil after COVID-19. Nat. Med. 2021, 27, 1629–1635.
DOI Google Scholar
[32]

Yang, Y.; Yang, M. H.; Yuan, J.; Wang, F. X.; Wang, Z. Q.; Li, J. X.; Zhang, M. X.; Xing, L.; Wei, J. L.; Peng, L. et al. Laboratory diagnosis and monitoring the viral shedding of SARS-CoV-2 infection. Innovation 2020, 1, 100061.
DOI Google Scholar
[33]

Dinnes, J.; Deeks, J. J.; Berhane, S.; Taylor, M.; Adriano, A.; Davenport, C.; Dittrich, S.; Emperador, D.; Takwoingi, Y.; Cunningham, J. et al. Rapid, point-of-care antigen and molecular-based tests for diagnosis of SARS-CoV-2 infection. Cochrane Database Syst. Rev. 2021, 2021, CD013705.
DOI Google Scholar
[34]

Kang, S. S.; Yang, M.; Hong, Z. S.; Zhang, L. P.; Huang, Z. X.; Chen, X. X.; He, S. H.; Zhou, Z. L.; Zhou, Z. C.; Chen, Q. Y. et al. Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites. Acta Pharm. Sin. B 2020, 10, 1228–1238.
DOI Google Scholar
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Publication history
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Publication history

Received: 22 April 2022
Revised: 01 July 2022
Accepted: 03 July 2022
Published: 17 August 2022
Issue date: April 2023

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