Ratiometric fluorescence immunoassay of SARS-CoV-2 nucleocapsid protein via Si-FITC nanoprobe-based inner filter effect
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.
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Graphical Abstract
The p-nitrophenyl (pNP) produced by the alkaline phosphatase (ALP)-mediated hydrolysis of p-nitrophenyl phosphate (pNPP) could quench Si fluorescence in Si-fluorescein isothiocyanate nanoparticles (FITC NPs) via the inner filter effect. In enzyme-linked immunosorbent assay (ELISA), an immunocomplex was formed by the recognition of capture antibody/nucleocapsid (N) protein/reporter antibody, and ALP-linked secondary antibody bound to the reporter antibody and induced pNPP hydrolysis to specifically quench Si fluorescence in Si-FITC NPs.
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.
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References
[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.
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.
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 Nano2018, 12, 7232–7238.
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.
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.
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.
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. Interfaces2019, 11, 16822–16829.
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.
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.
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. Interfaces2014, 6, 21270–21278.
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.
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.
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.
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.
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.
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.
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.
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. Nanoscale2020, 12, 19383–19389.
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.
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.
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. Interfaces2022, 14, 138–149.
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.
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. Acta2019, 1047, 208–213.
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.
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. B2018, 122, 12131–12138.
Vu, H. P.; Moreau, J. W. Thiocyanate adsorption on ferrihydrite and its fate during ferrihydrite transformation to hematite and goethite. Chemosphere2015, 119, 987–993.
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.
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. Innovation2020, 1, 100061.
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. B2020, 10, 1228–1238.
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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