Detection of hyaluronidase activity using fluorescence lifetime correlation spectroscopy to separate diffusing species and eliminate autofluorescence

Ryan M. Rich, Mark Mummert, Zygmunt Gryczynski, Julian Borejdo, Ignacy Gryczynski, Thomas J. Sørensen, Bo W. Laursen, Rafal Fudala

Research output: Chapter in Book/Report/Conference proceedingConference contributionpeer-review

4 Scopus citations


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.

Original languageEnglish
Title of host publicationSingle Molecule Spectroscopy and Superresolution Imaging VI
StatePublished - 28 May 2013
EventSingle Molecule Spectroscopy and Superresolution Imaging VI - San Francisco, CA, United States
Duration: 2 Feb 20133 Feb 2013

Publication series

NameProgress in Biomedical Optics and Imaging - Proceedings of SPIE
ISSN (Print)1605-7422


OtherSingle Molecule Spectroscopy and Superresolution Imaging VI
Country/TerritoryUnited States
CitySan Francisco, CA


  • Fluorescence
  • Fluorescence Correlation Spectroscopy
  • Hyaluronan
  • Time Correlated Single Photon Counting


Dive into the research topics of 'Detection of hyaluronidase activity using fluorescence lifetime correlation spectroscopy to separate diffusing species and eliminate autofluorescence'. Together they form a unique fingerprint.

Cite this