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

Rafal Fudala, Mark E. Mummert, Zygmunt Gryczynski, Ryan Rich, Julian Borejdo, Ignacy Gryczynski

Research output: Contribution to journalArticle

10 Scopus citations

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.

Original languageEnglish
Pages (from-to)69-73
Number of pages5
JournalJournal of Photochemistry and Photobiology B: Biology
Volume106
Issue number1
DOIs
StatePublished - 5 Jan 2012

Keywords

  • Fluorescence
  • Homo-FRET
  • Hyaluronan
  • Hyaluronidase
  • Lifetime based sensing

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