Full Citation: Fluorescence Analysis of Thermoresponsive Polymers. A.G. Ryder and C. Morris, Reviews in Fluorescence 2015 ,
Annual Volumes, Vol. 8, pp. 97-126, (2015). ISBN: 978-3-319-24607-9 (Hardcover), 978-3-319-24609-3 (ebook) Springer.
Page 15 of 31
both effects can be elucidated from the observed excitation wavelength dependence and a difference
in the fluorescence lifetimes of the N* and T* bands [74,100,106].
One example where fluorescence was used to assess the polarity change at temperatures near the
LCST exploited the solvatochromic shifts of the zwitterionic form of rhodamine X [107]. The
authors employed a rhodamine X labeled oligonucleotide composed of 25-mers of thymine (dT25ROX). In dioxane/water mixtures this probe shows a linear relationship between ET(30) and shifts
in the fluorescence emission maximum (in wavenumbers) for the ET(30) range between 50 and 65
Kcal.mol
–1
. They used this probe to look at the polarity changes of the PNIPAm shell of
PMMA/PNIPAm core-shell latex particles at temperatures near the LCST. For this system, the
calculated ET(30) value for the PNIPAm shell decreases in a sigmoidal manner as the temperature
increases from 15 to 45 °C. At the lower temperatures, the PNIPAm shell polarity is nearly identical
to that of water, while above the transition it is equivalent to the polarity of a dioxane/water mixture
(30% (v/v)). In contrast to the smooth transition observed in the wavelength shifts, the lifetime of
the dT25-ROX probe showed a sharp drop at the LCST which is ascribed to the refractive index change,
which accompanies the dehydration process during the phase change.
Derivatives of quinoxaline (N-(2,3-dimorpholinoquinoxalin-6-yl)acrylamide, QxA and N-(1-(2,3dimorpholinoquinoxalin-6-ylamino)prop-2-yl)methacrylamide, QxAlaMA) have also been
incorporated into PNIPAm [108]. These fluorophores when incorporated into the polymer showed
intense solvatochromism in their fluorescence without perturbing the LCST. The wavelength at the
maximum fluorescence intensity of the QxAlaMA-labeled PNIPAm dramatically blue-shifted (by ~20
nm) and the fluorescence intensity of the QxA-labeled PNIPAm significantly increased, by a factor of
~10, as the temperature increased from 30 to 34 °C.
5.4
CRITICAL MICELLE CONCENTRATION (CMC) AND AGGREGATION
MEASUREMENTS.
The CMC is a critical parameter that affects the macroscopic behavior of these thermoresponsive
polymers. Measuring the CMC is thus an important facet of polymer science and can usually be
achieved by conventional means using techniques like Dynamic Light Scattering (DLS). However,
in some cases the CMC can be low and as such is not very amenable to conventional measurement
methods. For example, the CMC of some amphiphilic block copolymers is of the order of ~10
–6
M
and cannot be easily determined by scattering methods. This problem can be overcome by using
single molecule detection (SMD) methods like fluorescence correlation spectroscopy (FCS) where one
can easily observe the very low concentration regime [109-111]. One straightforward method
collects FCS data from samples which have a fixed, very low concentration (typically 50 nM or less)
of a poorly water soluble fluorophore such as R6G in aqueous solutions of the thermoresponsive
polymer with varying concentrations (micro-molar range). The polymer concentrations need to be
Annual Volumes, Vol. 8, pp. 97-126, (2015). ISBN: 978-3-319-24607-9 (Hardcover), 978-3-319-24609-3 (ebook) Springer.
Page 15 of 31
both effects can be elucidated from the observed excitation wavelength dependence and a difference
in the fluorescence lifetimes of the N* and T* bands [74,100,106].
One example where fluorescence was used to assess the polarity change at temperatures near the
LCST exploited the solvatochromic shifts of the zwitterionic form of rhodamine X [107]. The
authors employed a rhodamine X labeled oligonucleotide composed of 25-mers of thymine (dT25ROX). In dioxane/water mixtures this probe shows a linear relationship between ET(30) and shifts
in the fluorescence emission maximum (in wavenumbers) for the ET(30) range between 50 and 65
Kcal.mol
–1
. They used this probe to look at the polarity changes of the PNIPAm shell of
PMMA/PNIPAm core-shell latex particles at temperatures near the LCST. For this system, the
calculated ET(30) value for the PNIPAm shell decreases in a sigmoidal manner as the temperature
increases from 15 to 45 °C. At the lower temperatures, the PNIPAm shell polarity is nearly identical
to that of water, while above the transition it is equivalent to the polarity of a dioxane/water mixture
(30% (v/v)). In contrast to the smooth transition observed in the wavelength shifts, the lifetime of
the dT25-ROX probe showed a sharp drop at the LCST which is ascribed to the refractive index change,
which accompanies the dehydration process during the phase change.
Derivatives of quinoxaline (N-(2,3-dimorpholinoquinoxalin-6-yl)acrylamide, QxA and N-(1-(2,3dimorpholinoquinoxalin-6-ylamino)prop-2-yl)methacrylamide, QxAlaMA) have also been
incorporated into PNIPAm [108]. These fluorophores when incorporated into the polymer showed
intense solvatochromism in their fluorescence without perturbing the LCST. The wavelength at the
maximum fluorescence intensity of the QxAlaMA-labeled PNIPAm dramatically blue-shifted (by ~20
nm) and the fluorescence intensity of the QxA-labeled PNIPAm significantly increased, by a factor of
~10, as the temperature increased from 30 to 34 °C.
5.4
CRITICAL MICELLE CONCENTRATION (CMC) AND AGGREGATION
MEASUREMENTS.
The CMC is a critical parameter that affects the macroscopic behavior of these thermoresponsive
polymers. Measuring the CMC is thus an important facet of polymer science and can usually be
achieved by conventional means using techniques like Dynamic Light Scattering (DLS). However,
in some cases the CMC can be low and as such is not very amenable to conventional measurement
methods. For example, the CMC of some amphiphilic block copolymers is of the order of ~10
–6
M
and cannot be easily determined by scattering methods. This problem can be overcome by using
single molecule detection (SMD) methods like fluorescence correlation spectroscopy (FCS) where one
can easily observe the very low concentration regime [109-111]. One straightforward method
collects FCS data from samples which have a fixed, very low concentration (typically 50 nM or less)
of a poorly water soluble fluorophore such as R6G in aqueous solutions of the thermoresponsive
polymer with varying concentrations (micro-molar range). The polymer concentrations need to be