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 16 of 31
selected such that they span a range above and below the suspected CMC value. At polymer
concentrations below the CMC, the fluorophore should remain dissolved in water, and only the
diffusion of free dye should be observed (free R6G has a hydrodynamic radius of 0.8 nm) [112]. As
the polymer concentration increase above the CMC value, an increasingly significant fraction of the
fluorophores will become associated with the micelles as they are formed. This association process
should result in the observation of an additional slow diffusion process being incorporated in the FCS
correlation curve.
For example, Adelselberger and co-workers used FCS to study the behavior of amphiphilic,
symmetric tri-block thermoresponsive copolymers having short, deuterated polystyrene (PS) end
blocks and a large PNIPAm middle block in aqueous solutions at very low concentration close to the
CMC [111]. Using FCS they found that at polymer concentrations above 0.9 μM, a second, slower
decay appeared in the correlation curves, indicating the onset of micelle formation. Fitting of the
correlation curves yielded hydrodynamic radii of 20.8 ± 0.7 nm for PS11-b-PNIPAm280- b-PS11 and
25.8 ± 0.9 nm for PS11-b-PNIPAm370-- b-PS11. These values were validated from DLS measurements
made at dilute (0.1 mg/mL) concentrations. The authors do however, point out that with these free
probes, micelles may still be present at very low concentrations (< 0.9 μM), but that there may be too
few to solubilize ample Rh6G molecules to generate sufficient signal that can be extracted from the
correlation curves. A variation of this methodology is to use polymer covalently labeled with a
fluorophore and measure the change in hydrodynamic radii via FCS [109,110]. This can be a very
sensitive method for determining the hydrodynamic radii of not only the micelles but also the unimers,
and intermediate, unstable aggregates.
While these SMD methods are elegant, care needs to be taken with interpretation of results because
of complications induced by the use of very low fluorophore and polymer concentrations. These
include changes in polymer and probe concentrations due to surface binding to the container walls,
which can be different for the free polymer and the various polymer assemblies. Consideration also
needs to be given to variations in fluorophore photophysics (quantum yield, lifetime etc.) generated by
the different physical environments of the solution and the micelle [113].
An extension of the FCS methodology was used by Wang and co-workers to study lateral diffusion
on PNIPAm brushes attached to solid surfaces [114]. They used a poly(2-vinylpyridine) probe
molecule that was covalently labeled with Alexa 488. Their studies enable the extraction of friction
force data and also a better understanding of how the PNIPAm brushes interact with polyelectrolyte
probe molecules. They showed that below the LCST, the decrease of viscosity of the solvent water
brought about a decrease in the friction forces via coupling of the lateral probe diffusion with the
motion of the brush chain. The LCST transition induced stiffening of the PNIPAm chain and the
hardening of the PNIPAm brushes above the LCST generated a large increase in friction forces.
Another study compared the diffusion of probe molecules interacting with
octadecyltriethoxysilane (OTE) monolayers and surface-tethered PNIPAm polymer chains of varying
thickness and surface coverage [115]. An interesting observation from this study was that the R6G
fluorophore interacted more strongly with PNIPAm compared to the OTE monolayer. This indicates
Annual Volumes, Vol. 8, pp. 97-126, (2015). ISBN: 978-3-319-24607-9 (Hardcover), 978-3-319-24609-3 (ebook) Springer.
Page 16 of 31
selected such that they span a range above and below the suspected CMC value. At polymer
concentrations below the CMC, the fluorophore should remain dissolved in water, and only the
diffusion of free dye should be observed (free R6G has a hydrodynamic radius of 0.8 nm) [112]. As
the polymer concentration increase above the CMC value, an increasingly significant fraction of the
fluorophores will become associated with the micelles as they are formed. This association process
should result in the observation of an additional slow diffusion process being incorporated in the FCS
correlation curve.
For example, Adelselberger and co-workers used FCS to study the behavior of amphiphilic,
symmetric tri-block thermoresponsive copolymers having short, deuterated polystyrene (PS) end
blocks and a large PNIPAm middle block in aqueous solutions at very low concentration close to the
CMC [111]. Using FCS they found that at polymer concentrations above 0.9 μM, a second, slower
decay appeared in the correlation curves, indicating the onset of micelle formation. Fitting of the
correlation curves yielded hydrodynamic radii of 20.8 ± 0.7 nm for PS11-b-PNIPAm280- b-PS11 and
25.8 ± 0.9 nm for PS11-b-PNIPAm370-- b-PS11. These values were validated from DLS measurements
made at dilute (0.1 mg/mL) concentrations. The authors do however, point out that with these free
probes, micelles may still be present at very low concentrations (< 0.9 μM), but that there may be too
few to solubilize ample Rh6G molecules to generate sufficient signal that can be extracted from the
correlation curves. A variation of this methodology is to use polymer covalently labeled with a
fluorophore and measure the change in hydrodynamic radii via FCS [109,110]. This can be a very
sensitive method for determining the hydrodynamic radii of not only the micelles but also the unimers,
and intermediate, unstable aggregates.
While these SMD methods are elegant, care needs to be taken with interpretation of results because
of complications induced by the use of very low fluorophore and polymer concentrations. These
include changes in polymer and probe concentrations due to surface binding to the container walls,
which can be different for the free polymer and the various polymer assemblies. Consideration also
needs to be given to variations in fluorophore photophysics (quantum yield, lifetime etc.) generated by
the different physical environments of the solution and the micelle [113].
An extension of the FCS methodology was used by Wang and co-workers to study lateral diffusion
on PNIPAm brushes attached to solid surfaces [114]. They used a poly(2-vinylpyridine) probe
molecule that was covalently labeled with Alexa 488. Their studies enable the extraction of friction
force data and also a better understanding of how the PNIPAm brushes interact with polyelectrolyte
probe molecules. They showed that below the LCST, the decrease of viscosity of the solvent water
brought about a decrease in the friction forces via coupling of the lateral probe diffusion with the
motion of the brush chain. The LCST transition induced stiffening of the PNIPAm chain and the
hardening of the PNIPAm brushes above the LCST generated a large increase in friction forces.
Another study compared the diffusion of probe molecules interacting with
octadecyltriethoxysilane (OTE) monolayers and surface-tethered PNIPAm polymer chains of varying
thickness and surface coverage [115]. An interesting observation from this study was that the R6G
fluorophore interacted more strongly with PNIPAm compared to the OTE monolayer. This indicates