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J Photochem Photobiol B. 2012 Jan 5;106:69-73. doi: 10.1016/j.jphotobiol.2011.10.005. Epub 2011 Oct 20.

Lifetime-based sensing of the hyaluronidase using fluorescein labeled hyaluronic acid.

Author information

  • 1Department of Molecular Biology and Immunology, Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center, Fort Worth, TX 76107, USA. Rafal.Fudala@unthsc.edu

Abstract

In this report we propose a lifetime-based sensing (LBS) for the detection of hyaluronidase (HA-ase). First, we heavily label hyaluronan macromolecules (HAs) with fluorescein amine. The fluorescein labeled HA (HA-Fl) has a weak fluorescence and short fluorescence lifetime due to an efficient self-quenching. Upon the addition of HA-ase, the brightness and lifetime of the sample increase. The cleavage of an HA macromolecule reduces the energy migration between fluorescein molecules and the degree of the self-quenching. A first order of the cleavage reaction depends on the amount of the HA-ase enzyme. We describe an HA-ase sensing strategy based on the lifetime changes of the fluorescein labeled HA in the presence of HA-ase. We demonstrate that the calibration of the sensing response is the same for the average lifetime as for a single exponential decay approximation, which significantly simplifies the analysis of the sensing measurements.

Published by Elsevier B.V.

PMID:
22082776
[PubMed - indexed for MEDLINE]
PMCID:
PMC3242895
Free PMC Article

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