Park, Juhwan Park, Minjun Kim, Junbeom Heo, Youhee Han, Bo Hoon Choi, Nakwon Park, Chulmin Lee, Raeseok Lee, Dong-Gun Chung, Seok Kang, Ji Yoon 2024-01-19T09:05:12Z 2024-01-19T09:05:12Z 2023-06-01 2023-07 0956-5663 https://pubs.kist.re.kr/handle/201004/113559 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. English Pergamon Press Ltd. Beads- and oil-free single molecule assay with immuno-rolling circle amplification for detection of SARS-CoV-2 from saliva Article 10.1016/j.bios.2023.115316 1 Biosensors and Bioelectronics, v.232 Biosensors and Bioelectronics 232 Y scie scopus 000985955500001 2-s2.0-85152598625 Biophysics Biotechnology & Applied Microbiology Chemistry, Analytical Electrochemistry Nanoscience & Nanotechnology Biophysics Biotechnology & Applied Microbiology Chemistry Electrochemistry Science & Technology - Other Topics Article IMMUNOSORBENT PROTEIN COVID-19 ELISA N protein Rolling circle amplification Saliva SARS-CoV-2