title: Lifetime-based sensing of the hyaluronidase using fluorescein labeled hyaluronic acid.
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.
authors.affiliation: Department 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
authors.forename: Rafal
authors.initials: R
authors.lastname: Fudala
authors.forename: Mark E
authors.initials: ME
authors.lastname: Mummert
authors.forename: Zygmunt
authors.initials: Z
authors.lastname: Gryczynski
authors.forename: Ryan
authors.initials: R
authors.lastname: Rich
authors.forename: Julian
authors.initials: J
authors.lastname: Borejdo
authors.forename: Ignacy
authors.initials: I
authors.lastname: Gryczynski
mesh_terms: D015374:Biosensing Techniques; D019793:Fluorescein / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D006820:Hyaluronic Acid / Q000737:chemistry*; D006821:Hyaluronoglucosaminidase / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D013997:Time Factors
chemical_list: D006820:Hyaluronic Acid; D006821:Hyaluronoglucosaminidase; D019793:Fluorescein
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