박민준 (한국과학기술연구원, 고려대학교) | 한빛사논문 > 한빛사

한빛사논문

조회330

박민준Minjun Park

한국과학기술연구원, 고려대학교

Biosens. Bioelectron., Volume 232, 15 July 2023, 115316 | https://doi.org/10.1016/j.bios.2023.115316 Beads-and oil-free single molecule assay with immuno-rolling circle amplification for detection of SARS-CoV-2 from saliva

Juhwan Park ^a,1, Minjun Park^a,e,1, Junbeom Kim^a, Youhee Heo ^a, Bo Hoon Han ^a, Nakwon Choi ^a, Chulmin Park ^b, Raeseok Lee ^b,c, Dong-Gun Lee ^b,c, Seok Chung ^e, Ji Yoon Kang^a,d

^aBrain Science Institute, Korea Institute of Science and Technology (KIST), Seoul, 02792, Republic of Korea

^bVaccine Bio Research Institute, College of Medicine, The Catholic University of Korea, Seoul, 16591, Republic of Korea

^cDivision of Infectious Diseases, Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, 16591, Republic of Korea

^dDivision of Bio-Medical Science and Technology, University of Science and Technology, Daejeon, Republic of Korea

^eSchool of Mechanical Engineering, Korea University, Seoul, Republic of Korea

¹These authors contributed equally to this work.

Corresponding author : Ji Yoon Kang

Abstract

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.

논문정보

형식Research article
게재일2023년 07월 (BRIC 등록일 2023-12-22)
연구진국내 연구진
분야 바이오·의료융합 > 바이오센싱 및 나노바이오물질

댓글 작성 로그인

CV 열람하기

연락처 보기