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                            University of North Texas Health Science Center
UNTHSC Scholarly Repository
	5-1-2016
Sensing and imaging of hyaluronidase activity using a long-lived fluorophore
	Rahul Chib
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University of North Texas Health Science Center
UNTHSC Scholarly Repository

Theses and Dissertations

5-1-2016

Sensing and imaging of hyaluronidase activity using
a long-lived fluorophore
Rahul Chib
University of North Texas Health Science Center at Fort Worth, [email protected]

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SENSING AND IMAGING OF HYALURONIDASE ACTIVITY USING A LONG-LIVED
FLUOROPHORE





DISSERTATION




Presented to the Graduate Council of the
Graduate School of Biomedical Sciences





University of North Texas
Health Science Center at Fort Worth




In Partial Fulfillment of the Requirements




For the Degree of
Doctor of Philosophy





By
Rahul Chib

Fort Worth, Texas
April 2016

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83








Table 3: Analysis of N-(-butanoic acid)-azatriangulenium tetrafluoroborate (ADOTA)

anisotropy decay using multiexponential model of on Silica thin film and PVA film.



3.5.Time resolved anisotropy

Time resolved anisotropy measurements presented in Figs. 8 and 9 shows some interesting

information. As expected in the case of the PVA matrix, initial anisotropy was high (0.3) with a

very long correlation time (>500 ns) (correlation time >> fluorescence lifetime) at both

observation wavelengths (560 and 620 nm). However, in case of silica gel matrix the recovered

initial anisotropy values are 0.21 and 0.19 for 560 and 620 nm observation and the measured

correlation times and rINF values are not significantly different (Table 3). The heterogeneous

nature of the correlation times is due to the populations of the dyes in different types of matrix

environment. Therefore, it will not be surprising if a part of the dye population is immobilized

completely and a part of it has a moderate freedom than the others. It is suggested in the

literature that the hydrogen bonding among silica mesh and the dye molecules undergoes

reshuffling in excited state and it may give rise to such correlation times. Moreover, the

estimated steady state anisotropy values at these wavelengths are 0.069 and 0.042. The drop in

the initial recovered anisotropy, in case of anisotropy decay, and steady state anisotropy values

suggests energy migration (if the dye molecules are sitting together below 40 A0 distances which

is the approximate Forster distance for such homo-transfer and is a possibility we cannot ignore).

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Although energy migration is a rapid process which takes place over the lifetime of the

fluorophore despite having a relatively long fluorescence lifetime in both matrices.



4. Conclusions

In the present work, the organic fluorophore, N-(u-butanoic acid)-azatriangulenium

tetrafluoroborate (ADOTA) was successfully incorporated into silica thin films by the sol-gel

process as well as in PVA films. The PVA matrix was chosen to study fluorescence properties of

ADOTA in a more rigid environment. Silica matrix provides an unusual environment for the

entrapped fluorophores and affects the dye properties in many different ways. A red shift of 15

nm was observed in the emission spectra of ADOTA in silica thin film. In addition, the

fluorescence lifetime of ADOTA at 560 nm observation in silica matrix was 12 ns compared to

20 ns in PVA film. However, when the wavelength of observation was placed at 620 nm, the

fluorescence lifetimes of ADOTA entrapped in both the matrices are almost similar. The

Lorentzian lifetime distribution shows the presence of broad distribution of the emitting species

in silica thin layer compared to PVA film. To sum up, considering all the observations such as

lower fluorescence efficiencies, shorter lifetimes and concentration dependent red shifted

emission suggests aggregation of dye molecules in the excited state. Ultrafast spectroscopy

experiments will further support our observations.

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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.

Compliance with ethical standards

Conflict of interest The authors declare that they have no competing
interests.

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