The COVID-19 crisis requires fast and highly sensitive tests for the early stage detection of the SARS-CoV-2 virus. For detecting the nucleocapsid protein (N protein), the most abundant viral antigen, we have employed upconversion nanoparticles that emit short-wavelength light under near-infrared excitation (976 nm). The anti-Stokes emission avoids autofluorescence and light scattering and thus enables measurements without optical background interference. The sandwich upconversion-linked immunosorbent assay (ULISA) can be operated both in a conventional analog mode and in a digital mode based on counting individual immune complexes. We have investigated how different antibody combinations affect the detection of the wildtype N protein and the detection of SARS-CoV-2 (alpha variant) in lysed culture fluid via the N protein. The ULISA yielded a limit of detection (LOD) of 1.3 pg/mL (27 fM) for N protein detection independent of the analog or digital readout, which is approximately 3 orders of magnitude more sensitive than conventional enzyme-linked immunosorbent assays or commercial lateral flow assays for home testing. In the case of SARS-CoV-2, the digital ULISA additionally improved the LOD by a factor of 10 compared to the analog readout.

CEITEC Central European Institute of Technology Masaryk University 625 00 Brno Czech Republic

Department of Biochemistry Faculty of Science Masaryk University 625 00 Brno Czech Republic

Department of Rare Earths Faculty of Chemistry Adam Mickiewicz University Poznań 61614 Poznań Poland

Institute of Analytical Chemistry Chemo and Biosensors University of Regensburg 93053 Regensburg Germany

Institute of Analytical Chemistry of the Czech Academy of Sciences 602 00 Brno Czech Republic

Zobrazit více v PubMed

Perez-Reche F. J.; Forbes K. J.; Strachan N. J. C. Importance of untested infectious individuals for interventions to suppress COVID-19. Sci. Rep. 2021, 11 (1), 20728.10.1038/s41598-021-00056-5. PubMed DOI PMC

Fröberg J.; Gillard J.; Philipsen R.; Lanke K.; Rust J.; van Tuijl D.; Teelen K.; Bousema T.; Simonetti E.; van der Gaast-de Jongh C. E.; Bos M.; van Kuppeveld F. J.; Bosch B. J.; Nabuurs-Franssen M.; van der Geest-Blankert N.; van Daal C.; Huynen M. A.; de Jonge M. I.; Diavatopoulos D. A. SARS-CoV-2 mucosal antibody development and persistence and their relation to viral load and COVID-19 symptoms. Nat. Commun. 2021, 12 (1), 5621.10.1038/s41467-021-25949-x. PubMed DOI PMC

Lee J.; Song J. U.; Shim S. R. Comparing the diagnostic accuracy of rapid antigen detection tests to real time polymerase chain reaction in the diagnosis of SARS-CoV-2 infection: A systematic review and meta-analysis. J. Clin. Virol. 2021, 144, 104985.10.1016/j.jcv.2021.104985. PubMed DOI PMC

van den Beld M. J. C.; Murk J. L.; Kluytmans J.; Koopmans M. P. G.; Reimerink J.; van Loo I. H. M.; Wegdam-Blans M. C. A.; Zaaijer H.; GeurtsvanKessel C.; Reusken C. Increasing the efficiency of a national laboratory response to COVID-19: a nationwide multicenter evaluation of 47 commercial SARS-CoV-2 immunoassays by 41 laboratories. J. Clin. Microbiol. 2021, 59 (9), e007672110.1128/JCM.00767-21. PubMed DOI PMC

Li X. W.; Xiong M. Y.; Deng Q. L.; Guo X. B.; Li Y. R. The utility of SARS-CoV-2 nucleocapsid protein in laboratory diagnosis. J. Clin. Lab. Anal. 2022, 36 (7), e2453410.1002/jcla.24534. PubMed DOI PMC

Liotti F. M.; Menchinelli G.; Lalle E.; Palucci I.; Marchetti S.; Colavita F.; La Sorda M.; Sberna G.; Bordi L.; Sanguinetti M.; Cattani P.; Capobianchi M. R.; Posteraro B. Performance of a novel diagnostic assay for rapid SARS-CoV-2 antigen detection in nasopharynx samples. Clin. Microbio. Infect. 2021, 27 (3), 487–488. 10.1016/j.cmi.2020.09.030. PubMed DOI PMC

Kabay G.; DeCastro J.; Altay A.; Smith K.; Lu H. W.; Capossela A. M.; Moarefian M.; Aran K.; Dincer C. Emerging biosensing technologies for the diagnostics of viral infectious diseases. Adv. Mater. 2022, 34 (30), 2201085.10.1002/adma.202201085. PubMed DOI

Yuan H.; Chen P.; Wan C.; Li Y.; Liu B. F. Merging microfluidics with luminescence immunoassays for urgent point-of-care diagnostics of COVID-19. Trends Anal. Chem. 2022, 157, 116814.10.1016/j.trac.2022.116814. PubMed DOI PMC

Resch-Genger U.; Gorris H. H. Perspectives and challenges of photon-upconversion nanoparticles - Part I: routes to brighter particles and quantitative spectroscopic studies. Anal. Bioanal. Chem. 2017, 409 (25), 5855–5874. 10.1007/s00216-017-0499-z. PubMed DOI

Gorris H. H.; Resch-Genger U. Perspectives and challenges of photon-upconversion nanoparticles - Part II: bioanalytical applications. Anal. Bioanal. Chem. 2017, 409 (25), 5875–5890. 10.1007/s00216-017-0482-8. PubMed DOI

Mickert M. J.; Farka Z.; Kostiv U.; Hlaváček A.; Horák D.; Skládal P.; Gorris H. H. Measurement of sub-femtomolar concentrations of prostate-specific antigen through single-molecule counting with an upconversion-linked immunosorbent assay. Anal. Chem. 2019, 91 (15), 9435–9441. 10.1021/acs.analchem.9b02872. PubMed DOI

Sedlmeier A.; Hlaváček A.; Birner L.; Mickert M. J.; Muhr V.; Hirsch T.; Corstjens P. L. A. M.; Tanke H. J.; Soukka T.; Gorris H. H. Highly sensitive laser scanning of photon-upconverting nanoparticles on a macroscopic scale. Anal. Chem. 2016, 88 (3), 1835–1841. 10.1021/acs.analchem.5b04147. PubMed DOI

Hlaváček A.; Farka Z.; Mickert M. J.; Kostiv U.; Brandmeier J. C.; Horák D.; Skládal P.; Foret F.; Gorris H. H. Bioconjugates of photon-upconversion nanoparticles for cancer biomarker detection and imaging. Nat. Prot. 2022, 17 (4), 1028–1072. 10.1038/s41596-021-00670-7. PubMed DOI

Farka Z.; Mickert M. J.; Pastucha M.; Mikušová Z.; Skládal P.; Gorris H. H. Advances in optical single-molecule detection: En route to supersensitive bioaffinity assays. Angew. Chem. Int. Edit 2020, 59 (27), 10746–10773. 10.1002/anie.201913924. PubMed DOI PMC

Gorris H. H.; Soukka T. What digital immunoassays can learn from ambient analyte theory: A perspective. Anal. Chem. 2022, 94 (16), 6073–6083. 10.1021/acs.analchem.1c05591. PubMed DOI

Brandmeier J. C.; Raiko K.; Farka Z.; Peltomaa R.; Mickert M. J.; Hlaváček A.; Skládal P.; Soukka T.; Gorris H. H. Effect of particle size and surface chemistry of photon-upconversion nanoparticles on analog and digital immunoassays for cardiac troponin. Adv. Healthc. Mater. 2021, 10 (18), 2100506.10.1002/adhm.202100506. PubMed DOI PMC

Alexaki K.; Kyriazi M. E.; Greening J.; Taemaitree L.; El-Sagheer A. H.; Brown T.; Zhang X. L.; Muskens O. L.; Kanaras A. G. A SARS-CoV-2 sensor based on upconversion nanoparticles and graphene oxide. RSC Adv. 2022, 12 (29), 18445–18449. 10.1039/D2RA03599E. PubMed DOI PMC

Balinski B.Australian-made COVID-19 test returns results within minutes. Create. https://createdigital.org.au/australian-made-covid-19-test-results-within-minutes/.

Makhneva E.; Sklenárová D.; Brandmeier J. C.; Hlaváček A.; Gorris H. H.; Skládal P.; Farka Z. Influence of Label and Solid Support on the Performance of Heterogeneous Immunoassays. Anal. Chem. 2022, 94 (47), 16376–16383. 10.1021/acs.analchem.2c03543. PubMed DOI

Ogata A. F.; Maley A. M.; Wu C.; Gilboa T.; Norman M.; Lazarovits R.; Mao C. P.; Newton G.; Chang M.; Nguyen K.; Kamkaew M.; Zhu Q.; Gibson T. E.; Ryan E. T.; Charles R. C.; Marasco W. A.; Walt D. R. Ultra-sensitive serial profiling of SARS-CoV-2 antigens and antibodies in plasma to understand disease progression in COVID-19 patients with severe disease. Clin. Chem. 2020, 66 (12), 1562–1572. 10.1093/clinchem/hvaa213. PubMed DOI PMC

Lahtinen S.; Lyytikäinen A.; Päkkilä H.; Hömppi E.; Perälä N.; Lastusaari M.; Soukka T. Disintegration of hexagonal NaYF4:Yb3+,Er3+ upconverting nanoparticles in aqueous media: The role of fluoride in solubility equilibrium. J. Phys. Chem. C 2017, 121 (1), 656–665. 10.1021/acs.jpcc.6b09301. DOI

Boom R.; Sol C. J. A.; Salimans M. M. M.; Jansen C. L.; Wertheimvandillen P. M. E.; Vandernoordaa J. Rapid and simple method for purification of nucleic-acids. J. Clin. Microbiol. 1990, 28 (3), 495–503. 10.1128/jcm.28.3.495-503.1990. PubMed DOI PMC

Shatzkes K.; Teferedegne B.; Murata H. A simple, inexpensive method for preparing cell lysates suitable for downstream reverse transcription quantitative PCR. Sci. Rep. 2014, 4, 4659.10.1038/srep04659. PubMed DOI PMC

HyTest SARS-CoV-2 antibodies and detection of variants. HyTest. https://shop.hytest.fi/spree/products/4156/SARS-CoV-2_detection_of_variants.pdf?1648709822.

Hytest TechNotes: Reagents of SARS-CoV-2 antigen and antibody assays. HyTest. https://hytest.fi/sites/5cd13840ff4f702c0cbc4c8d/content_entry5cd13897ff4f702c0cbc4cb2/5f09b34cff4f703a3f35bdf3/files/SARS-CoV-2_TechNotes.pdf?1647263232.

Rissin D. M.; Kan C. W.; Campbell T. G.; Howes S. C.; Fournier D. R.; Song L.; Piech T.; Patel P. P.; Chang L.; Rivnak A. J.; Ferrell E. P.; Randall J. D.; Provuncher G. K.; Walt D. R.; Duffy D. C. Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nat. Biotechnol. 2010, 28 (6), 595–599. 10.1038/nbt.1641. PubMed DOI PMC

Białobrzeska W.; Ficek M.; Dec B.ło.; Osella S.; Trzaskowski B.; Jaramillo-Botero A.; Pierpaoli M.; Rycewicz M.ł; Dashkevich Y.; Łega T.; Malinowska N.; Cebula Z.; Bigus D.; Firganek D.; Biega E.; Dziabowska K.; Brodowski M.; Kowalski M.; Panasiuk M.ła.; Gromadzka B.; Zołedowska S.; Nidzworski D.; Pyrc K.; Goddard W. A.; Bogdanowicz R. Performance of electrochemical immunoassays for clinical diagnostics of SARS-CoV-2 based on selective nucleocapsid N protein detection: Boron-doped diamond, gold and glassy carbon evaluation. Biosens. Bioelectron. 2022, 209, 114222.10.1016/j.bios.2022.114222. PubMed DOI PMC

Pollock N. R.; Savage T. J.; Wardell H.; Lee R. A.; Mathew A.; Stengelin M.; Sigal G. B. Correlation of SARS-CoV-2 nucleocapsid antigen and RNA concentrations in nasopharyngeal samples from children and adults using an ultrasensitive and quantitative antigen assay. J. Clin. Microbiol. 2021, 59 (4), e03077-2010.1128/JCM.03077-20. PubMed DOI PMC

Cai Q.; Mu J.; Lei Y.; Ge J.; Aryee A. A.; Zhang X.; Li Z. Simultaneous detection of the spike and nucleocapsid proteins from SARS-CoV-2 based on ultrasensitive single molecule assays. Anal. Bioanal. Chem. 2021, 413 (18), 4645–4654. 10.1007/s00216-021-03435-z. PubMed DOI PMC

Park J. H.; Lee G. Y.; Song Z.; Bong J. H.; Chang Y. W.; Cho S.; Kang M. J.; Pyun J. C. Capacitive biosensor based on vertically paired electrodes for the detection of SARS-CoV-2. Biosens. Bioelectron. 2022, 202, 113975.10.1016/j.bios.2022.113975. PubMed DOI PMC

Grant B. D.; Anderson C. E.; Williford J. R.; Alonzo L. F.; Glukhova V. A.; Boyle D. S.; Weigl B. H.; Nichols K. P. SARS-CoV-2 coronavirus nucleocapsid antigen-detecting half-strip Lateral Flow Assay toward the development of point of care tests using commercially available reagents. Anal. Chem. 2020, 92 (16), 11305–11309. 10.1021/acs.analchem.0c01975. PubMed DOI

Grant B. D.; Anderson C. E.; Alonzo L. F.; Garing S. H.; Williford J. R.; Baughman T. A.; Rivera R.; Glukhova V. A.; Boyle D. S.; Dewan P. K.; Weigl B. H.; Nichols K. P. A SARS-CoV-2 coronavirus nucleocapsid protein antigen-detecting lateral flow assay. PLoS One 2021, 16 (11), e025881910.1371/journal.pone.0258819. PubMed DOI PMC

Farka Z.; Mickert M. J.; Hlaváček A.; Skládal P.; Gorris H. H. Single molecule upconversion-linked immunosorbent assay with extended dynamic range for the sensitive detection of diagnostic biomarkers. Anal. Chem. 2017, 89 (21), 11825–11830. 10.1021/acs.analchem.7b03542. PubMed DOI

Magnetic microbead-based upconversion immunoassay with laser-induced breakdown spectroscopy readout for the detection of prostate-specific antigen

Upconversion Nanoparticle-Based Dot-Blot Immunoassay for Quantitative Biomarker Detection

Sandwich Immuno-RCA Assay with Single Molecule Counting Readout: The Importance of Biointerface Design

Bright photon upconversion in LiYbF4:Tm3+@LiYF4 nanoparticles and their application for singlet oxygen generation and in immunoassay for SARS-CoV-2 nucleoprotein