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.
Highlights
Immunoassays are a unique and well-established methodology for the detection of disease biomarkers
Cardiac troponin I is a typical biomarker used for early diagnosis of acute myocardial infarction, which is crucial for prompt treatment [1]
Proteins can be labeled with fluorophores by conjugation, which enables their use in imaging, cell sorting, and biosensing [4]
Summary
Based on the Relaxation of Quenching, Exemplified by Detection of Cardiac Troponin I. Seung-Wan Kim 1,† , Il-Hoon Cho 2,† , Ji-Na Park 1 , Sung-Min Seo 1 and Se-Hwan Paek 1,3, *.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have