Dye-Doped Polymeric Particles for Sensing and Imaging - Advanced Fluorescence Reporters in Chemistry and Biology - page 205

be ground to fine particles ( several micrometer). Examples include poly(4- tert -

butyl styrene) and Ormosils. Some less brittle materials can be ground at cryogenic
temperatures although it is often problematic to obtain particles of the desired size.

3.6 Purification of the Beads

Purification is often required for the beads obtained by the techniques described
above since undesired substances such as surfactants, coupling agents, etc. need
to be removed. This is also valid for dye molecules noncovalently adsorbed on
the surface of the beads since they usually have different properties (sensitivity,
cross-talk to other analytes, leaching, etc.) compared to the molecules located in the
core. The dye-doped beads can be purified by repeated precipitation which is
achieved by adding salts (typically sodium chloride). In certain cases (typically
for large beads) the addition of salts is not necessary so that the beads can be
isolated by centrifugation. Washing with ethanol often helps remove lipophilic dye
molecules adsorbed on the surface provided that the polymer is not swellable.
Alternatively, dialysis can be useful especially if a hydrophilic water-soluble
indicator is covalently coupled to the bead surface.

4 Optical Read-Out Schemes

Most dye-doped beads are interrogated using luminescent methods since they enable
higher sensitivity compared to absorptiometry and reflectometry. Luminescence
intensity is still the most popular analytical parameter due to the simplicity of the
method. Obviously, luminescence intensity is not referenced if determined at a single
wavelength and is affected by drifts of the opto-electronic system, light scattering,
turbidity of the sample, etc. [ 20 ]. Therefore, ratiometric measurements at two differ-
ent wavelengths are widely used [ 21 ]. Different techniques for wavelength-ratio-
metric sensing involving bot single dyes and responsive-irresponsive dye pairs have
been recently reviewed [ 128 ]. In one of the applied techniques an inert dye (insensi-
tive to the analyte of interest and other species) is coembedded into the beads to be
excited simultaneously with the indicator. F¨rster resonance energy transfer (FRET)
is often employed to enable referenced measurements (where the intensities from a
FRET donor and a FRET acceptor are collected at two different wavelengths).
Fluorescein-rhodamine pairs are the most popular for designing FRET nanosensors
for pH. As demonstrated recently for planar optodes [ 22 ], FRET can also be used to
increase sensor brightness by means of light harvesting. This principle was also used
to enhance the brightness of dye-doped polymeric beads [ 17 ].
Measurement of luminescence decay time represents another method of self-
referencing. The method is widely used for the indicators that possess luminescence
decay times in the microsecond and millisecond domain which can be interrogated