Fluorescence detection of hyaluronidase

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19 Scopus citations

Abstract

We labeled hyaluronan (HA) with two fluorophores, fluorescein amine and rhodamine B amine. These two fluorophores are suitable for a fluorescence (Foerster) resonance energy transfer (FRET) which results in a fluorescein quenching and an enhanced rhodamine emission. Such labeled HA (HA-FRET) is a potential sensor for HA degradation. We studied fluorescence properties of HA-FRET in the absence and presence of hyaluronidase enzyme (HA-ase). The time-resolved fluorescence measurements indicate more than 50% of FRET in the absence of HA-ase. In the presence of HA-ase FRET decreases with time, and relative fluorescence intensities of fluorescein and rhodamine shifts to fluorescein indicating a release of FRET. The kinetics of the digestion process of HA by HA-ase depends on the concentration of the enzyme. We demonstrate that simultaneous measurements of green and red emission of HA-FRET can be used in ratio metric detection of the HA-ase presence and activity. This in turn, can be utilized for the construction of a robust but reliable HA-ase sensing device.

Original languageEnglish
Pages (from-to)473-477
Number of pages5
JournalJournal of Photochemistry and Photobiology B: Biology
Volume104
Issue number3
DOIs
StatePublished - 2 Sep 2011

Keywords

  • FRET
  • Fluorescence
  • Hyaluronan
  • Hyaluronidase
  • Ratio metric sensing

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