Dye-Doped Polymeric Particles for Sensing and Imaging - Advanced Fluorescence Reporters in Chemistry and Biology - page 209

from the oxygen indicator is observed. Residual blue luminescence from poly-
fluorene enables ratiometric imaging of oxygen tension with optimal sensitivity
( I 0 / I

3.7). Additionally, the nanobeads were found to be nicely suitable for oxygen
imaging under two-photon excitation which is not surprising since polyfluorens were
previously found to have very high two photon absorption cross-sections [ 32 ]. The
authors also demonstrated suitability of the nanobeads for intracellular imaging.
Pfister et al. [ 33 ] used difluoroboron dibenzoylmethane conjugate with poly
(lactic acid) to prepare biocompatible oxygen-sensing nanoparticles ( Ø 100 nm).
The polymer-dye conjugate was found to possess both blue fluorescence and green
phosphorescence. This unique property, in principle, enables ratiometric sensing of
oxygen with one indicator only. However, the phosphorescence decay time was
found to be too long ( 200 ms) for optimal sensitivity in biological systems. The
decay time for the iodine-substituted difluoroboron dibenzoylmethane-poly(lactic
acid) conjugate was found to decrease by tenfold (

¼

4.82 ms) due to the heavy
atom effect [ 34 ]. This modified material was demonstrated to be suitable for
ratiometric imaging of tumor hypoxia (pO 2 <

t 0 ¼

1 kPa).

5.1.2 Beads for Sensing and Imaging of pH

Cross-linked polyacrylamide beads are often the material of choice for preparation
of optical pH nanosensors [ 35 ]. A pH indicator (usually a fluorescein derivative)
and a reference dye are introduced prior to polymerization and are physically
entrapped into the beads. Such nanosensor operates fully reversibly and show
acceptable photostability. However, leaching of the hydrophilic indicators often
compromises the performance of such materials. For example, as much as 40% of
the indicator leached out within the first 2 days [ 35 ]. Sun et al. [ 36 ] attempted to
minimize leaching of an indicator (fluorescein) and a reference dye (rhodamine) by
covalently immobilizing them into the polyacrylamide network. Although leaching
was substantially reduced compared to the dyes simply dissolved in the polymer
(6% vs. 70% in 6 h) the extent of leaching was still not negligible. Allard and
Larpent [ 37 ] minimized leaching by covalently attaching fluorescein isothiocyanate
to the surface of amino-functionalized crosslinked polystyrene nanobeads ( Ø 20 nm).
No detectable leaching was observed for at least 1 week. A highly hydrophobic
reference dye 9,10-diphenylanthracene was also embedded into the beads by swelling.
However, the reference dye required excitation in the UV region (375 nm) which is
not very practical.
McNamara et al. [ 38 ] developed water-dispersible microbeads for ratiometric
imaging of pH. The 1.6 m polystyrene beads were covered with a phospholipid

layer containing covalently coupled fluorescein and rhodamine. The material was
suitable for sensing pH in bulk solution and in macrophages.
Funfak et al. [ 39 ] demonstrated recently that pH-sensitive microbeads based on
8-hydroxypyrene-1,3,6-trisulfonate covalently attached to amino-functionalized
poly(hydroxyethylmethacrylate) ( Ø 2.5

m

m) can be successfully used to monitor

m