Twisted perylene dyes enable highly fluorescent and photostable nanoparticles

Article abstract of DOI:10.1039/b815507k

Polymeric fluorescent nanoparticles with covalently embedded perylene fluorophores were developed by facile synthesis strategy and their advanced features of extremely high fluorescence intensity, non-photoblinking and excellent photostability were experi

Full text of DOI:10.1039/b815507k

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Twisted perylene dyes enable highly fluorescent and photostable
nanoparticlesw
Zhiyuan Tian,z Andrew D. Shallerz and Alexander D. Q. Li*
Received (in Austin, TX, USA) 5th September 2008, Accepted 10th October 2008
First published as an Advance Article on the web 13th November 2008
DOI: 10.1039/b815507k
Polymeric fluorescent nanoparticles with covalently embedded
perylene fluorophores were developed by facile synthesis strategy
and their advanced features of extremely high fluorescence
intensity, non-photoblinking and excellent photostability were
experimentally confirmed at the single nanoparticle level.
Fluorescent nanoparticles (FNPs) have attracted considerable
interest for their wide range of emerging applications in live
cell imaging, biosensing, and optoelectronic devices.1 Practical
FNPs, especially for biological applications, require high
fluorescent intensity, photostability, biocompatibility and
relatively small diameter (o50 nm). Among currently avail-
Scheme
1 Polymeric nanoparticle synthesis with twisted PDI
able FNPs, inorganic semiconductor quantum dots possess
brightness up to 20 times brighter than a single molecule,
desired small diameter and improved photostability but suffer
from photoblinking and cyto-toxicity.2–4 Dye-doped polymer
nanoparticles are limited by fluorophore aggregation and self-
quenching as well as dye leaching.5 Additionally, fluorescent
intensity rarely scales linearly with the number of dyes
imbedded in FNPs due to obstacles such as singlet-singlet
annihilation, self-quenching and Stern–Volmer quenching.
Thus, despite reported enhanced brightness calculated from
ensemble measurements,6 experimental data for FNPs with
diameter o50 nm and fluorescence 420 times brighter than a
single dye are not reported.7,8 A relatively unexplored alter-
native is the use of FNPs with covalently bound fluorophores.
Covalent attachment limits chromophore mobility and dis-
perses the monomers within FNPs which potentially reduces
fluorophore aggregation and therefore fluorescence self-
quenching, providing a distinct advantage over doping. In this
communication, we have developed photostable and bio-
compatible polymeric nanoparticles with covalently bound
perylene dye possessing over 50 times brighter fluorescence
as compared to single-dye molecules such as rhodamine 6G.
Because the tetrachloro-perylene diimide (PDI) derivative has
four substituted chlorine atoms in the bay positions to twist the
molecule out of plane (371), it maintains high fluorescent
quantum yield (Ffl B 0.9) and high photostability.9 The highly
twisted structure frustrates p–p stacking, reducing self- or colli-
sional quenching within the tight confines of a FNP, thus a
strategy to fabricate bright FNP. The twisted PDI was mono-
benzoylated and functionalized with an acryloyl moiety to form
monomer.
the monomer 1 which is shown in Scheme 1. Our approach to
constructing polymer FNPs here is based on a modified emul-
sion polymerization method10 with the twisted PDIs covalently
embedded within the hydrophobic cavities of polymeric nano-
particles. Acrylamide and styrene were polymerized with minor
amounts of functional monomers, including optically active
perylene dye, the cross-linker divinyl benzene, and butyl acrylate
for lowering the glass transition temperature of the nano-
particles. Several batches were identically prepared except for
varying feed dye concentration, ranging from 0.3 to 2.4 wt%.
These nanoparticles are easily suspended in water due to their
hydrophilic shell, enabling feasible application in live cell experi-
ments. Because the nanoparticle fluorescence originates from
multiple dispersed dyes, one nanoparticle shines 450 times
brighter than a single dye. Furthermore, no perylene dye loss
was found for the aqueous nanoparticle samples after washing
with organic solvent because the PDI components are covalently
anchored inside the nanoparticle. This is an advantage over
dye-doped nanoparticles, in which dye molecules may aggregate
or leach into the environment such as a live cell.
The absorption and emission spectra for an aqueous nano-
particle sample exhibit typical spectral characteristics com-
pared to the monomer (Fig. 1(a)) indicating minimal perylene
aggregation, thus contributing to their bright fluorescence.
Additionally, the FNP solutions have Ffl E 0.5, further
evidence of minimal stacking interactions. Both the absor-
bance and fluorescence lmax are slightly red-shifted from the
monomer due to differences in local environments (styrene vs.
CH2Cl2). The nanoparticle emission vibronic bands are also
slightly broadened due to non-homogenous broadening. FNP
size was measured by both TEM and dynamic light scattering
(DLS).8 TEM, the standard measurement method, indicated
an average diameter of 40 nm (Fig. 1(b)).
Department of Chemistry, Washington State University, Pullman, WA
99164, USA. E-mail: dequan@wsu.edu; Fax: (509)-335-8867;
Tel: (509)-335-7196
w Electronic supplementary information (ESI) available: Materials,
methods and supporting figures. See DOI: 10.1039/b815507k
z These two authors contributed equally to this work.
While various methods and calculations have been used to
estimate single particle brightness from ensemble measurements,
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180 | Chem. Commun., 2009, 180–182

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brightness occurrence averaged over several slide samples
demonstrate an unambiguous increase in brightness distribu-
tion between single nanoparticles and single PDIs. Also,
with increasing PDI concentration, the FNP brightness
scales approximately linearly, indicating dye incorporation is
proportional to feed ratio over this concentration range
(0.3–2.4 w/w%).
Why are these nanoparticles so bright compared to previous
examples of dye-doped nanoparticles? First, covalent attach-
ment allows a large number of dyes to incorporate without
changing the nanoparticle structure. Second, even in the case
of the highest PDI feed concentration, minimal spectral
features of dye aggregation were observed. Thus, nearly all
of the dyes retain their monomeric status following poly-
merization. Apparently, the fluorescence self-quenching is
largely suppressed, likely due to frustration of p–p stacking
by the twisted PDI structure and the spacing effect of other
co-monomers. Third, the core–shell structure effectively
encapsulates most fluorophores and reduces photobleaching
by molecular oxygen.
Fig. 1 (a) Normalized ensemble absorbance and fluorescence spectra
of an aqueous nanoparticle colloid (solid blue) compared to the
monomer in CH2Cl2 (green dash) and the fluorescence from a single
nanoparticle (red). (b) TEM image exhibiting average particle size =
40 nm. The aggregation on TEM grids is an artifact caused by drying,
as dynamic light scattering reveals no aggregation in solution.
the most accurate method to truly determine single particle
brightness is by single particle experiments.8 Thus, using a single
molecule set-up as described in the ESI,w we performed wide-
field and diffraction limited measurements of single particle
brightness.
To further probe the detailed photodynamic properties of
the FNPs, fluorescence time traces of single nanoparticle from
each batch were probed by diffraction-limited excitation and
the fluorescence collected by avalanche photo diode (APD) as
shown in Fig. 3. For comparison, a typical time trace of the
monomer dye is also shown (insert). As typical for a single
molecule, the monomer displays a sharp on-and-off process
(photo-blinking). In sharp contrast, the fluorescence time trace
for individual nanoparticles exhibit distinctively different
fluorescence with a gradual decay due to multiple dye mole-
cules within each nanoparticle. Immediately upon opening the
shutter, all nanoparticles show highly bright fluorescence (the
Fig. 2 reveals the brightness, determined by CCD wide-field
imaging, of single twisted PDI monomers and FNPs prepared
with varying feed dye amounts. Fig. 2(a) and (b) are shown at
the same color scale with unambiguous increase in brightness
of the nanoparticles over the monomer. Fig. 2(c)–(e) are at a
much higher color scale and show brightness increases with
increasing PDI feed concentration. Notably, many of the
nanoparticles in Fig. 2(e) fluoresce 480 times brighter than
a single fluorophore. Histograms generated from statistical
Fig. 2 (Top) Single molecule/particle wide-field CCD images (2 s integration time) for (a) monomer, (b) 0.3, (c) 0.6, (d) 1.2 and (e) 2.4 w/w%
samples; (a) and (b) are at the same color scale while (c)–(e) are at a much higher scale. In sample (e), several of the particles approach 80 times
brighter (I 4 8000) than a single monomer (I E 100). (Bottom) Histograms summarize the statistical brightness of each batch. While a single
monomer exhibits brightness of around 100, the nanoparticle samples (b)–(e) exhibit mean intensities 7, 19, 25 and 56 times brighter, respectively.
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In conclusion, we have successfully prepared polymeric
nanoparticles with high fluorescent brightness and excellent
photostability by covalently embedding a twisted perylene dye
into core–shell type polymeric nanoparticles. The high photo-
stability, quantum yield, and unique steric structure of the
perylene monomer, coupled with high monomer concentration
and core-shell encapsulation enabled by covalent attachment,
are believed to mainly contribute to the remarkable brightness
and improved photostability. These facile, ultrabright probes
ultimately advance the capability for practical applications
in live cell imaging, biosensing and in the development of
optoelectronic nanodevices.
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182 | Chem. Commun., 2009, 180–182