title: A High-Performance Fluorescence Immunoassay Based on the Relaxation of Quenching, Exemplified by Detection of Cardiac Troponin I.
abstract: The intramolecular fluorescence self-quenching phenomenon is a major drawback in developing high-performance fluorometric biosensors which use common fluorophores as signal generators. We propose two strategies involving liberation of the fluorescent molecules by means of enzymatic fragmentation of protein or dehybridization of double-stranded DNA. In the former, bovine serum albumin (BSA) was coupled with the fluorescent BODIPY dye (Red BSA), and then immobilized on a solid surface. When the insolubilized Red BSA was treated with proteinase K (10 units/mL) for 30 min, the fluorescent signal was significantly increased (3.5-fold) compared to the untreated control. In the second case, fluorophore-tagged DNA probes were linked to gold nanoparticles by hybridization with capture DNA strands densely immobilized on the surface. The quenched fluorescence signal was recovered (3.7-fold) by thermal dehybridization, which was induced with light of a specific wavelength (e.g., 530 nm) for less than 1 min. We next applied the Red BSA self-quenching relaxation technique employing enzymatic fragmentation to a high-performance immunoassay of cardiac troponin I (cTnI) in a microtiter plate format. The detection limit was 0.19 ng/mL cTnI, and the fluorescent signal was enhanced approximately 4.1-fold compared with the conventional method of direct measurement of the fluorescent signal from a non-fragmented fluorophore-labeled antibody.
authors.affiliation: Department of Bio-Microsystem Technology, Korea University, 145 Anam-ro, Sungbuk-gu, Seoul 02841, Korea. kimseungwan@korea.ac.kr.
authors.forename: Seung-Wan
authors.initials: SW
authors.lastname: Kim
authors.affiliation: Department of Biomedical Laboratory Science, College of Health Science, Eulji University, Seongnam 13135, Korea. chiuchun0716@gmail.com.
authors.forename: Il-Hoon
authors.initials: IH
authors.lastname: Cho
authors.affiliation: Department of Bio-Microsystem Technology, Korea University, 145 Anam-ro, Sungbuk-gu, Seoul 02841, Korea. gina4664@naver.com.
authors.forename: Ji-Na
authors.initials: JN
authors.lastname: Park
authors.affiliation: Department of Bio-Microsystem Technology, Korea University, 145 Anam-ro, Sungbuk-gu, Seoul 02841, Korea. ohyeh81@hotmail.com.
authors.forename: Sung-Min
authors.initials: SM
authors.lastname: Seo
authors.affiliation: Department of Bio-Microsystem Technology, Korea University, 145 Anam-ro, Sungbuk-gu, Seoul 02841, Korea. shpaek@korea.ac.kr.|Department of Biotechnology and Bioinformatics, Korea University, 2511 Sejong-ro, Sejong 30019, Korea. shpaek@korea.ac.kr.
authors.forename: Se-Hwan
authors.initials: SH
authors.lastname: Paek
mesh_terms: D015374:Biosensing Techniques; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D006046:Gold; D007118:Immunoassay; D019210:Troponin I / Q000032:analysis*
chemical_list: D005456:Fluorescent Dyes; D019210:Troponin I; D006046:Gold
keywords: enzymatic fragmentation; fluorescence quenching; immunoassay for cardiac troponin I; photothermal local heating; signal enhancement