Semiconducting polymer dots (Pdots)
are a type of conjugated
polymer
(CP) nanoparticle with highly advantageous fluorescence properties
and a growing breadth of applications in bioanalysis and imaging.
Exceptional brightness is a widely demonstrated advantage of Pdots.
Resistance to photobleaching (PB) is an often-touted advantage but
is far less studied. Here, we present a detailed study on the PB of
Pdots. The effects of the particle size, pH, oxygen level, and anti-fade
agents on PB were examined through ensemble and single-particle fluorescence
measurements for multiple Pdot compositions. Physical changes during
PB were assessed by nanoparticle tracking analysis, gel electrophoresis,
Förster resonance energy transfer, and other fluorescence quenching
experiments. Spectral shifts, increases in the fluorescence lifetime,
reductions in quantum yield, and changes in electrophoretic mobility
were some of the properties observed to vary upon PB. Loss of fluorescence
under continuous illumination was from formal bleaching and also from
the introduction of quenching mechanisms. Oxygen-dependent and oxygen-independent
mechanisms of PB were suggested by the data, with the balance of these
mechanisms dependent on both the CP and the illumination intensity.
The oxygen-independent mechanism appeared to be intrinsic to the volume
of the CP and may be limited by exciton–exciton annihilation
at high illumination intensity. The oxygen-dependent mechanism appeared
to bleach the Pdots from the surface of the particle inward, forming
a bleached shell, and introduced new functional groups. Overall, the
PB of Pdots was a complex process with photophysical and chemical
alterations, and homogeneous and heterogeneous effects, dependent
on particle properties and experimental conditions. Changes induced
by PB have implications for many biological applications of Pdots,
and the results of this study will help guide the development of Pdot
materials with greater resistance toward bleaching.