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Sensors 2016, 16(5), 669; doi:10.3390/s16050669

A High-Performance Fluorescence Immunoassay Based on the Relaxation of Quenching, Exemplified by Detection of Cardiac Troponin I

1
Department of Bio-Microsystem Technology, Korea University, 145 Anam-ro, Sungbuk-gu, Seoul 02841, Korea
2
Department of Biomedical Laboratory Science, College of Health Science, Eulji University, Seongnam 13135, Korea
3
Department of Biotechnology and Bioinformatics, Korea University, 2511 Sejong-ro, Sejong 30019, Korea
These authors equally contributed to this work.
*
Author to whom correspondence should be addressed.
Academic Editor: Alexander Star
Received: 25 March 2016 / Revised: 26 April 2016 / Accepted: 5 May 2016 / Published: 10 May 2016
(This article belongs to the Section Biosensors)
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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. View Full-Text
Keywords: signal enhancement; fluorescence quenching; enzymatic fragmentation; photothermal local heating; immunoassay for cardiac troponin I signal enhancement; fluorescence quenching; enzymatic fragmentation; photothermal local heating; immunoassay for cardiac troponin I
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Kim, S.-W.; Cho, I.-H.; Park, J.-N.; Seo, S.-M.; Paek, S.-H. A High-Performance Fluorescence Immunoassay Based on the Relaxation of Quenching, Exemplified by Detection of Cardiac Troponin I. Sensors 2016, 16, 669.

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