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 22 of 31
that the polymer LCST is directly affected by the K
+
ion concentration, increasing by ~9 °C as the K
+
concentration increased from 0 to 300 M. This reasonably rapid process (~4 second response time)
can be monitored by measuring the FRET efficiency calculated from the fluorescence intensity ratio
measured at 588 and 529 nm.
Many biomedical uses for PNIPAm involve the preparation of complex macro-, or meso-scale
structures and the assembly process can be studied using fluorescence. For example, the 4acrylamidofluorescein-modified poly(N-isopropylacrylamide-co-acrylic acid) (PNIPAm-co-AAc*)
was used with simple fluorescence microscopy to observe layer-by-layer (LbL) deposition of microgel
thin films [136]. The method is reasonably effective at showing coverage and layer formation, but
because of its non-confocal nature the resolution normal to the surface is very poor. Another area in
which fluorescence techniques can be useful for studying thermoresponsive polymers is in the LbL
assembly of polyelectrolyte multilayers on soft and porous PNIPAm microgels. One facet of the LbL
process is that the polyelectrolytes can interdigitate both with each other and with the microgel during
multilayer formation. The problem is further compounded by the fact that the particles are often submicron in size and thus not amenable to conventional microscopy evaluation. Using FCS, however,
one can easily distinguish between free, labeled polyelectrolytes and those that are bound to the
microgel. Wong and co-workers used dual color FCS to confirm that two different polyelectrolytes
were binding onto the same microgel particles (~400 nm in size) of PNIPAm [137].
One of the drawbacks with conventional FCS measurements is that one cannot generally get
absolute diffusion coefficients from the data and thus one has to correlate with standards of known
values. In dual-focus FCS (2f-FCS), one uses two focal volumes, which are a precisely known
distance apart and generate an overlapping detection volume. This enables accurate and precise
quantitative measurement of absolute diffusion coefficient values [138]. 2f-FCS measurements have
been undertaken at different temperatures to determine the hydrodynamic radii of bare nanogels
(p(NIPAM-co-AA-co-rhodamine)) and nanogels coated with various numbers of layers of
polyelectrolytes [139]. These temperature dependent studies showed that the polyelectrolyte
multilayer shell was still bound to the nanogel during the phase transition at the LCST.
6 CONCLUSIONS
Fluorescence spectroscopy offers a range of convenient methodologies for the analysis of
thermoresponsive polymers. The most widespread application is for the monitoring of the phase and
polarity changes at the LCST. The high sensitivity and low probe concentrations required ensures
that the fluorescence analysis has a minimal impact on polymer structure or physical behavior.
Furthermore, many of these analytical techniques can be performed using standard off-the-shelf,
inexpensive fluorescence spectrometers. Of increasing importance is the use of single molecule
detection methods to probe the dynamic processes that occur at very low polymer concentrations.
Annual Volumes, Vol. 8, pp. 97-126, (2015). ISBN: 978-3-319-24607-9 (Hardcover), 978-3-319-24609-3 (ebook) Springer.
Page 22 of 31
that the polymer LCST is directly affected by the K
+
ion concentration, increasing by ~9 °C as the K
+
concentration increased from 0 to 300 M. This reasonably rapid process (~4 second response time)
can be monitored by measuring the FRET efficiency calculated from the fluorescence intensity ratio
measured at 588 and 529 nm.
Many biomedical uses for PNIPAm involve the preparation of complex macro-, or meso-scale
structures and the assembly process can be studied using fluorescence. For example, the 4acrylamidofluorescein-modified poly(N-isopropylacrylamide-co-acrylic acid) (PNIPAm-co-AAc*)
was used with simple fluorescence microscopy to observe layer-by-layer (LbL) deposition of microgel
thin films [136]. The method is reasonably effective at showing coverage and layer formation, but
because of its non-confocal nature the resolution normal to the surface is very poor. Another area in
which fluorescence techniques can be useful for studying thermoresponsive polymers is in the LbL
assembly of polyelectrolyte multilayers on soft and porous PNIPAm microgels. One facet of the LbL
process is that the polyelectrolytes can interdigitate both with each other and with the microgel during
multilayer formation. The problem is further compounded by the fact that the particles are often submicron in size and thus not amenable to conventional microscopy evaluation. Using FCS, however,
one can easily distinguish between free, labeled polyelectrolytes and those that are bound to the
microgel. Wong and co-workers used dual color FCS to confirm that two different polyelectrolytes
were binding onto the same microgel particles (~400 nm in size) of PNIPAm [137].
One of the drawbacks with conventional FCS measurements is that one cannot generally get
absolute diffusion coefficients from the data and thus one has to correlate with standards of known
values. In dual-focus FCS (2f-FCS), one uses two focal volumes, which are a precisely known
distance apart and generate an overlapping detection volume. This enables accurate and precise
quantitative measurement of absolute diffusion coefficient values [138]. 2f-FCS measurements have
been undertaken at different temperatures to determine the hydrodynamic radii of bare nanogels
(p(NIPAM-co-AA-co-rhodamine)) and nanogels coated with various numbers of layers of
polyelectrolytes [139]. These temperature dependent studies showed that the polyelectrolyte
multilayer shell was still bound to the nanogel during the phase transition at the LCST.
6 CONCLUSIONS
Fluorescence spectroscopy offers a range of convenient methodologies for the analysis of
thermoresponsive polymers. The most widespread application is for the monitoring of the phase and
polarity changes at the LCST. The high sensitivity and low probe concentrations required ensures
that the fluorescence analysis has a minimal impact on polymer structure or physical behavior.
Furthermore, many of these analytical techniques can be performed using standard off-the-shelf,
inexpensive fluorescence spectrometers. Of increasing importance is the use of single molecule
detection methods to probe the dynamic processes that occur at very low polymer concentrations.