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Fluorescent biosensor for the detection of hyaluronidase: intensity-based ratiometric sensing and fluorescence lifetime-based sensing using a long lifetime azadioxatriangulenium (ADOTA) fluorophore

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Abstract

In this report, we have designed a rapid and sensitive, intensity-based ratiometric sensing as well as lifetime-based sensing probe for the detection of hyaluronidase activity. Hyaluronidase expression is known to be upregulated in various pathological conditions. We have developed a fluorescent probe by heavy labeling of hyaluronic acid with a new orange/red-emitting organic azadioxatriangulenium (ADOTA) fluorophore, which exhibits a long fluorescence lifetime (∼20 ns). The ADOTA fluorophore in water has a peak fluorescence lifetime of ∼20 ns and emission spectra centered at 560 nm. The heavily ADOTA-labeled hyaluronic acid (HA-ADOTA) shows a red shift in the peak emission wavelength (605 nm), a weak fluorescence signal, and a shorter fluorescence lifetime (∼4 ns) due to efficient self-quenching and formation of aggregates. In the presence of hyaluronidase, the brightness and fluorescence lifetime of the sample increase with a blue shift in the peak emission to its original wavelength at 560 nm. The ratio of the fluorescence intensity of the HA-ADOTA probe at 560 and 605 nm can be used as the sensing method for the detection of hyaluronidase. The cleavage of the hyaluronic acid macromolecule reduces the energy migration between ADOTA molecules, as well as the degree of self-quenching and aggregation. This probe can be efficiently used for both intensity-based ratiometric sensing as well as fluorescence lifetime-based sensing of hyaluronidase. The proposed method makes it a rapid and sensitive assay, useful for analyzing levels of hyaluronidase in relevant clinical samples like urine or plasma.

Scheme showing cleavage of HA-ADOTA probe by hyaluronidase and the change in the emission spectrum of HA-ADOTA probe before and after cleavage by hyaluronidase

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Acknowledgments

This work was supported by UNTHSC intramural grant RI6120 (R.F.), Sigma Xi grants in aid of research G20141015656984 (R.C.), UNTHSC pre-doctoral bridge grant RI6171 (R.C.), NIH grant R01EB12003 (Z.G.), and NSF grant CBET-1264608 (I.G.). We would like to thank Dr. Andras Lacko and Dr. Nirupama Sabnis for providing us the DU-145 cell line.

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Authors and Affiliations

  1. Department of Cell Biology, Immunology and Microbiology, Center for Fluorescence Technologies and Nanomedicine, University of North Texas Health Science Center, Fort Worth, TX, 76107, USA

    Rahul Chib, Sunil Shah, Ignacy Gryczynski, Julian Borejdo, Zygmunt Gryczynski & Rafal Fudala

  2. Mental Sciences Institute, University of North Texas Health Science Center, Fort Worth, TX, 76107, USA

    Mark Mummert

  3. Nano-Science Center & Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100, København Ø, Denmark

    Ilkay Bora & Bo W. Laursen

  4. Department of Physics and Astronomy, Texas Christian University, Fort Worth, TX, 76129, USA

    Robert Pendry & Zygmunt Gryczynski

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  1. Rahul Chib
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Chib, R., Mummert, M., Bora, I. et al. Fluorescent biosensor for the detection of hyaluronidase: intensity-based ratiometric sensing and fluorescence lifetime-based sensing using a long lifetime azadioxatriangulenium (ADOTA) fluorophore. Anal Bioanal Chem 408, 3811–3821 (2016). https://doi.org/10.1007/s00216-016-9472-5

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