Beads- and oil-free single molecule assay with immuno-rolling circle amplification for detection of SARS-CoV-2 from saliva

Juhwan Park, Minjun Park, Junbeom Kim, Youhee Heo, Bo Hoon Han, Nakwon Choi, Chulmin Park, Raeseok Lee, Dong Gun Lee, Seok Chung, Ji Yoon Kang

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)


Digital enzyme linked immunosorbent assays (ELISA) can be used to detect various antigens such as spike (S) or nucleocapsid (N) proteins of SARS-CoV-2, with much higher sensitivity compared to that achievable using conventional antigen tests. However, the use of microbeads and oil for compartmentalization in these assays limits their user-friendliness and causes loss of assay information due to the loss of beads during the process. To improve the sensitivity of antigen test, here, we developed an oil- and bead-free single molecule counting assay, with rolling circle amplification (RCA) on a substrate. With RCA, the signal is localized at the captured region of an antigen, and the signal from a single antigen molecule can be visualized using the same immune-reaction procedures as in the conventional ELISA. Substrate-based single molecule assay was theoretically evaluated for kd value, and the concentration of capture and detection antibodies. As a feasibility test, biotin-conjugated primer and mouse IgG conjugates were detected even at femto-molar concentrations with this digital immuno-RCA. Using this method, we detected the N protein of SARS-CoV-2 with a limit of detection less than 1 pg/mL more than 100-fold improvement compared to the detection using conventional ELISA. Furthermore, testing of saliva samples from COVID-19 patients and healthy controls (n = 50) indicated the applicability of the proposed method for detection of SARS-CoV-2 with 99.5% specificity and 90.9% sensitivity.

Original languageEnglish
Article number115316
JournalBiosensors and Bioelectronics
Publication statusPublished - 2023 Jul 15

Bibliographical note

Funding Information:
This research was supported mainly by the Korea Medical Device Development Fund grant funded by the Korean government (the Ministry of Science and ICT , the Ministry of Trade, Industry and Energy , the Ministry of Health & Welfare , the Ministry of Food and Drug Safety ) (KMDF_PR_20200901_0072), and by the Nano-Connect Technology Research Program through the National Research Foundation of Korea (NRF) , funded by the Ministry of Science and ICT (NRF- 2021M3H4A4079294 ).

Publisher Copyright:
© 2023 The Authors


  • COVID-19
  • N protein
  • Rolling circle amplification
  • Saliva
  • SARS-CoV-2

ASJC Scopus subject areas

  • Biotechnology
  • Biophysics
  • Biomedical Engineering
  • Electrochemistry


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