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Proceedings Paper

Detection of hyaluronidase activity using fluorescence lifetime correlation spectroscopy to separate diffusing species and eliminate autofluorescence
Author(s): Ryan M. Rich; Mark Mummert; Zygmunt Gryczynski; Julian Borejdo; Ignacy Gryczynski; Thomas Just Sørensen; Bo W. Laursen; Rafal Fudala
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Paper Abstract

The over-expression of hyaluronidase has been linked to many types of cancer, and thus we present here a technique for hyaluronidase detection and quantification using Fluorescence Correlation Spectroscopy (FCS). Our probe consists of Hyaluronan macromolecules (HAs) heavily loaded with fluorescein dye to the extent that the dye experiences self-quenching, and these HAs are detected as very bright, slowly moving particles by FCS. Hyaluronidase cleaves HAs into HA fragments, increasing the concentration of independent fluorescent molecules diffusing through the detection volume. The cleavage of HAs releases the self- quenching so that the intensity of emission is drastically increased. Both the concentration of fluorescent particles and intensity are measured simultaneously and correlated to the concentration of hyaluronidase. Also, our time correlated system allows us to assess the heterogeneity of the HA solution. Subpopulations of slowly moving particles with short-lived radiative decay may be separated from fast-moving particles of long-lived radiative decay and studied independently in a technique known as Fluorescence Lifetime Correlation Spectroscopy (FLCS). Further, we assess the use of the AzaDiOxaTriAngulenium (ADOTA) dye for FCS experiments. Its lifetime is significantly longer than that of the autofluorescence that plagues fluorescence experiments involving cells or tissue, and thus the fluorescence decay of the probe can be easily identified and separated from autofluorescence by FLCS. We demonstrate this by labeling HAs with ADOTA and adding free Rhodamine 123 to the solution to simulate the autofluorescence. We show that the combination of ADOTA and FLCS allow construction of an FCS-based hyaluronidase assay despite the presence of severe autofluorescence.

Paper Details

Date Published: 22 February 2013
PDF: 12 pages
Proc. SPIE 8590, Single Molecule Spectroscopy and Superresolution Imaging VI, 859003 (22 February 2013); doi: 10.1117/12.2007261
Show Author Affiliations
Ryan M. Rich, The Univ. of North Texas Health Science Ctr. (United States)
Mark Mummert, The Univ. of North Texas Health Science Ctr. (United States)
Zygmunt Gryczynski, The Univ. of North Texas Health Science Ctr. (United States)
Texas Christian Univ. (United States)
Julian Borejdo, The Univ. of North Texas Health Science Ctr. (United States)
Ignacy Gryczynski, The Univ. of North Texas Health Science Ctr. (United States)
Thomas Just Sørensen, The Univ. of Copenhagen (Denmark)
Bo W. Laursen, The Univ. of Copenhagen (Denmark)
Rafal Fudala, The Univ. of North Texas Health Science Ctr. (United States)

Published in SPIE Proceedings Vol. 8590:
Single Molecule Spectroscopy and Superresolution Imaging VI
Jörg Enderlein; Ingo Gregor; Zygmunt Karol Gryczynski; Rainer Erdmann; Felix Koberling, Editor(s)

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