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 contribution

4 Citations (Scopus)

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.

Original languageEnglish
Title of host publicationSingle Molecule Spectroscopy and Superresolution Imaging VI
DOIs
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
Volume8590
ISSN (Print)1605-7422

Other

OtherSingle Molecule Spectroscopy and Superresolution Imaging VI
CountryUnited States
CitySan Francisco, CA
Period2/02/133/02/13

Fingerprint

Hyaluronoglucosaminidase
Hyaluronic Acid
Spectrum Analysis
Macromolecules
macromolecules
Fluorescence
Spectroscopy
Fluorescence Spectrometry
life (durability)
fluorescence
spectroscopy
Coloring Agents
Dyes
dyes
Rhodamine 123
Quenching
decay
Plague
quenching
Fluorescein

Keywords

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

Cite this

Rich, R. M., Mummert, M., Gryczynski, Z., Borejdo, J., Gryczynski, I., Sørensen, T. J., ... Fudala, R. (2013). Detection of hyaluronidase activity using fluorescence lifetime correlation spectroscopy to separate diffusing species and eliminate autofluorescence. In Single Molecule Spectroscopy and Superresolution Imaging VI [859003] (Progress in Biomedical Optics and Imaging - Proceedings of SPIE; Vol. 8590). https://doi.org/10.1117/12.2007261
Rich, Ryan M. ; Mummert, Mark ; Gryczynski, Zygmunt ; Borejdo, Julian ; Gryczynski, Ignacy ; Sørensen, Thomas J. ; Laursen, Bo W. ; Fudala, Rafal. / Detection of hyaluronidase activity using fluorescence lifetime correlation spectroscopy to separate diffusing species and eliminate autofluorescence. Single Molecule Spectroscopy and Superresolution Imaging VI. 2013. (Progress in Biomedical Optics and Imaging - Proceedings of SPIE).
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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.",
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Rich, RM, Mummert, M, Gryczynski, Z, Borejdo, J, Gryczynski, I, Sørensen, TJ, Laursen, BW & Fudala, R 2013, Detection of hyaluronidase activity using fluorescence lifetime correlation spectroscopy to separate diffusing species and eliminate autofluorescence. in Single Molecule Spectroscopy and Superresolution Imaging VI., 859003, Progress in Biomedical Optics and Imaging - Proceedings of SPIE, vol. 8590, Single Molecule Spectroscopy and Superresolution Imaging VI, San Francisco, CA, United States, 2/02/13. https://doi.org/10.1117/12.2007261

Detection of hyaluronidase activity using fluorescence lifetime correlation spectroscopy to separate diffusing species and eliminate autofluorescence. / Rich, Ryan M.; Mummert, Mark; Gryczynski, Zygmunt; Borejdo, Julian; Gryczynski, Ignacy; Sørensen, Thomas J.; Laursen, Bo W.; Fudala, Rafal.

Single Molecule Spectroscopy and Superresolution Imaging VI. 2013. 859003 (Progress in Biomedical Optics and Imaging - Proceedings of SPIE; Vol. 8590).

Research output: Chapter in Book/Report/Conference proceedingConference contribution

TY - GEN

T1 - Detection of hyaluronidase activity using fluorescence lifetime correlation spectroscopy to separate diffusing species and eliminate autofluorescence

AU - Rich, Ryan M.

AU - Mummert, Mark

AU - Gryczynski, Zygmunt

AU - Borejdo, Julian

AU - Gryczynski, Ignacy

AU - Sørensen, Thomas J.

AU - Laursen, Bo W.

AU - Fudala, Rafal

PY - 2013/5/28

Y1 - 2013/5/28

N2 - 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.

AB - 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.

KW - Fluorescence

KW - Fluorescence Correlation Spectroscopy

KW - Hyaluronan

KW - Time Correlated Single Photon Counting

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U2 - 10.1117/12.2007261

DO - 10.1117/12.2007261

M3 - Conference contribution

AN - SCOPUS:84878073424

SN - 9780819493590

T3 - Progress in Biomedical Optics and Imaging - Proceedings of SPIE

BT - Single Molecule Spectroscopy and Superresolution Imaging VI

ER -

Rich RM, Mummert M, Gryczynski Z, Borejdo J, Gryczynski I, Sørensen TJ et al. Detection of hyaluronidase activity using fluorescence lifetime correlation spectroscopy to separate diffusing species and eliminate autofluorescence. In Single Molecule Spectroscopy and Superresolution Imaging VI. 2013. 859003. (Progress in Biomedical Optics and Imaging - Proceedings of SPIE). https://doi.org/10.1117/12.2007261