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 21 of 31
the phenomenon of cononsolvency exhibited by the PNIPAm/water/methanol ternary system. The
study involved both the PNIPAm-Da polymer and the cross-linked PNIPAm-DA gels [67]. The
swelling of these polymers and gels decreased abruptly in aqueous solutions containing 7-25 mol %
methanol and increased gradually in systems with a higher methanol concentration. Shifts in the
dansyl wavelength of maximum emission, changes in the fluorescence lifetimes, and changes in the
rotational diffusion coefficients could all be correlated with the macroscopic changes in swelling
volume.
In some cases, the swelling behavior of PNIPAm can be affected by the presence of other materials
in solution. When freely diffusing pyrene was used as a probe of PNIPAm behavior, the introduction
of urea changes in the pyrene emission only above the LCST [130]. This arose from urea induced
swelling of the PNIPAm compact coil conformation, caused by disruption of the intramolecular
hydrophobic interaction. This caused the pyrene probe to experience a much more hydrophobic
environment which was measured by the ratio of the emission intensities I3/I1. This study also
showed that the coil structure was reasonably robust and could be observed at urea concentrations of
up to 3M.
The swelling behavior of thermoresponsive polymers can also be investigated using Förster
Resonance Energy Transfer (FRET), where the degree of energy transfer provides information as to
separation between donor and acceptor fluorophores [59]. For example, Jones and co-workers used
FRET studies to analyze core-shell PNIPAm microgels (both the core and shell components were
lightly cross-linked with N,N´-methylene(bisacrylamide)), where the core was doubly labeled with
cyanine Cy5 (donor) and Cy5.5 (acceptor) [68]. In these structures, the PNIPAm shell can restrict
the core from swelling to its native volume, and thus the extent of core expansion will be a function of
shell thickness. To covalently attach the fluorophores, the core contained a small percentage of amine
groups for post-polymerization modification with the cyanine fluorophores, which were functionalized
with N-hydroxysuccinimidyl ester. For the naked core, the degree of FRET was low when it was
swollen to its maximum volume below the LCST (31 °C). The presence of a PNIPAm shell produces
a significant degree of FRET under the same solution conditions, indicating that the polymer chains
are more constrained relative to the fully swollen state. By monitoring the degree of energy transfer
in the absence and presence of the shell, over a range of temperature values, the researchers observed
the decreased swelling ability of the core in the presence of the added shell, and were thus able to
estimate the shell thickness. This FRET method has obvious advantages when compared to
conventional Photon Correlation Spectroscopy (PCS) measurements, which can only yield an apparent
particle size, and not discriminate between changes in shell thickness and core compression associated
with thicker shells.
The combination of thermoresponsive polymers and the FRET methodology has also been
exploited for sensing applications. In one such example, PNIPAm microgels were modified to
incorporate potassium ion recognizing 4-acrylamidobenzo18-crown-6 residues (B18C6Am) and then
a FRET pair of fluorophores (4-(2-acryloyloxyethylamino) -7-nitro-2,1,3-benzoxadiazole (NBDAE),
and rhodamine-B-based FRET acceptors (RhBEA)) [135]. The key operational feature is the fact
Annual Volumes, Vol. 8, pp. 97-126, (2015). ISBN: 978-3-319-24607-9 (Hardcover), 978-3-319-24609-3 (ebook) Springer.
Page 21 of 31
the phenomenon of cononsolvency exhibited by the PNIPAm/water/methanol ternary system. The
study involved both the PNIPAm-Da polymer and the cross-linked PNIPAm-DA gels [67]. The
swelling of these polymers and gels decreased abruptly in aqueous solutions containing 7-25 mol %
methanol and increased gradually in systems with a higher methanol concentration. Shifts in the
dansyl wavelength of maximum emission, changes in the fluorescence lifetimes, and changes in the
rotational diffusion coefficients could all be correlated with the macroscopic changes in swelling
volume.
In some cases, the swelling behavior of PNIPAm can be affected by the presence of other materials
in solution. When freely diffusing pyrene was used as a probe of PNIPAm behavior, the introduction
of urea changes in the pyrene emission only above the LCST [130]. This arose from urea induced
swelling of the PNIPAm compact coil conformation, caused by disruption of the intramolecular
hydrophobic interaction. This caused the pyrene probe to experience a much more hydrophobic
environment which was measured by the ratio of the emission intensities I3/I1. This study also
showed that the coil structure was reasonably robust and could be observed at urea concentrations of
up to 3M.
The swelling behavior of thermoresponsive polymers can also be investigated using Förster
Resonance Energy Transfer (FRET), where the degree of energy transfer provides information as to
separation between donor and acceptor fluorophores [59]. For example, Jones and co-workers used
FRET studies to analyze core-shell PNIPAm microgels (both the core and shell components were
lightly cross-linked with N,N´-methylene(bisacrylamide)), where the core was doubly labeled with
cyanine Cy5 (donor) and Cy5.5 (acceptor) [68]. In these structures, the PNIPAm shell can restrict
the core from swelling to its native volume, and thus the extent of core expansion will be a function of
shell thickness. To covalently attach the fluorophores, the core contained a small percentage of amine
groups for post-polymerization modification with the cyanine fluorophores, which were functionalized
with N-hydroxysuccinimidyl ester. For the naked core, the degree of FRET was low when it was
swollen to its maximum volume below the LCST (31 °C). The presence of a PNIPAm shell produces
a significant degree of FRET under the same solution conditions, indicating that the polymer chains
are more constrained relative to the fully swollen state. By monitoring the degree of energy transfer
in the absence and presence of the shell, over a range of temperature values, the researchers observed
the decreased swelling ability of the core in the presence of the added shell, and were thus able to
estimate the shell thickness. This FRET method has obvious advantages when compared to
conventional Photon Correlation Spectroscopy (PCS) measurements, which can only yield an apparent
particle size, and not discriminate between changes in shell thickness and core compression associated
with thicker shells.
The combination of thermoresponsive polymers and the FRET methodology has also been
exploited for sensing applications. In one such example, PNIPAm microgels were modified to
incorporate potassium ion recognizing 4-acrylamidobenzo18-crown-6 residues (B18C6Am) and then
a FRET pair of fluorophores (4-(2-acryloyloxyethylamino) -7-nitro-2,1,3-benzoxadiazole (NBDAE),
and rhodamine-B-based FRET acceptors (RhBEA)) [135]. The key operational feature is the fact