One-pot production of fluorescent surface-labeled polymeric nanoparticles via miniemulsion polymerization with bodipy surfmers

Article abstract of DOI:10.1021/ma300090a

New molecules combining the functionalities of surface activity, polymerizability, and fluorescent properties within one molecule that could be seen as a fluorescent surfmer (surfactant and monomer) were successfully synthesized. A long hydrocarbon tail c

Full text of DOI:10.1021/ma300090a

Article
pubs.acs.org/Macromolecules
One-Pot Production of Fluorescent Surface-Labeled Polymeric
Nanoparticles via Miniemulsion Polymerization with Bodipy
Surfmers
Rudiger Sauer,† Andrey Turshatov,*,† Stanislav Baluschev,†,‡ and Katharina Landfester†
̈
†Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany
‡Optics and Spectroscopy Department, Faculty of Physics, Sofia University “St. Kliment Ochridski”, 5 James Bourchier, 1164 Sofia,
Bulgaria
ABSTRACT: New molecules combining the functionalities of
surface activity, polymerizability, and fluorescent properties
within one molecule that could be seen as a fluorescent
surfmer (surfactant and monomer) were successfully synthe-
sized. A long hydrocarbon tail capped with a methacrylamide
unit was anchored to a mono- or double-sulfonated Bodipy
core. Time-resolved fluorescence measurements in aqueous
solutions of the dyes enable one to trace the micelle formation
and to find strong polarity dependence of fluorescence
properties of the dyes. By using these molecules, polystyrene
nanoparticles with fluorescent interfaces were synthesized via
miniemulsion polymerization. The surfmers can be used alone
or as surfactant additional to sodium dodecyl sulfate that provides stable emulsions and dispersions with a wide range (100−250
nm) of realizable particle sizes after polymerization. All synthesized nanoparticles showed bright fluorescence and can be easily
investigated with conventional fluorescent confocal microscopy. Only the fluorescence of particles with double-sulfonated
surfmer used is strongly sensitive to fluorescent quenchers as sodium iodide or methyl viologene dissolved in the continuous
phase. In contrast, the fluorescence of nanoparticles labeled with the monosulfonated surfmer is only weakly quenched that might
be explained by specific orientation of the Bodipy core at particle/water interface.
phosphonic acids,8 oligo(ethylene glycol)s,9−12 sulfobetains,13
INTRODUCTION
Fluorescence is a powerful tool to gain further insight into a
widespread field of material interactions; beneficial is the
■
and sulfonated peptides.14 Furthermore, a small number of
amphiphilic molecules were synthesized to gain insight into
lipid and membrane systems and reactions or dynamics in
noninvasive and nondestructive nature of the measurements for
associated systems.15−18 Additionally, sensor systems are
the sample. In recent years there have been an abundance of
applications in the interdisciplinary field between chemistry,
physics, and medicine. For this reason a large variety of
fluorescent labels and probes were realized. One promising
class of stable fluorophors is the dipyrromethene boron
difluoride (Bodipy) derivatives due to their outstanding
properties, such as high quantum yields, excellent thermal-,
photo-, and chemical-stability, as well as good resistance toward
becoming the focus of interest to trace ions19−21 or pH22
values for example in cells.
In this area often nanoparticle systems come into play, as
they have several advantages. The first one is the possibility to
have several dyes in one particle for ratiometric labeling;
another point is the problem that several dye classes tend to be
cytotoxic in their free form.23 Additionally, fluorescent
nanoparticles show a much higher fluorescent signal than
aggregation, and additionally a lot of sides are accessible for
fluorescent molecules that are used for labeling.24 As the dye is
entrapped in the particle, the local concentration is much
higher, so that the absolute amount of dye needed for detection
is reduced. A major problem in the field of particle systems is
the mobility of the dye; often the dyes are physically entrapped
in the polymeric bulk, either by encapsulation in the synthesis
of the particle25 or by swelling and soaking in of a dye solution
into the particle.26,27 Depending on the hydrophilicity of the
chemical modification.1,2 They were first synthesized by Treibs
and Kreuzer in 1968.3 Bodipy’s show narrow absorption and
emission bands; this is especially interesting for applications
where more than one dye has to be employed and
superposition has to be prevented, which is often the case
especially when dealing with cells and cellular uptake. For the
application in biological systems water-soluble dyes are
necessary. The first water-soluble Bodipy was introduced by
Wories et al.4 by sulfonation of the 2- and 6-position of the
Bodipy core; they were able to show that this Bodipy was
nontoxic in rats. Only a few more water-soluble Bodipy
derivatives were realized ever since, bearing carboxylic acids,5−7
Received: January 13, 2012
Revised: March 29, 2012
© XXXX American Chemical Society
A
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Article
dye and the swellability of the polymeric material, leaching of
the dye could be pronounced and reach up to 50% within 48
h.28
Herein the synthesis and application of two new surfmers are
presented that are bearing, to the best of our knowledge, for the
first time a Bodipy label in the headgroup, as an additional
surfactant or sole surfactant in miniemulsion polymerization,
for a fluorescent decoration of nanoparticle surfaces. The proof
of copolymerization was conducted by HPLC measurements
and the location of the surfmer at the surface of the particles by
variation in fluorescent lifetime in the presence of a fluorescent
quencher.
Another approach is to covalently attach the label to the
particle, either by copolymerizing a dye with a polymerizable
function27 or by covalently attaching a dye to a functionalized
particle surface.29 The latter has the advantage that the dye is in
direct contact to the surrounding media, and neither interaction
with the polymeric bulk material that might change slightly the
photophysical behavior of the dye nor aggregation of the dye
within the polymerization of the monomer could occur.
Additionally, in the case where sensor systems are monitored,
the analyte has no need to diffuse into the particle, which
prevents a potential reduction of the analyte concentration at
the interaction side. Disadvantageous is the need for coupling
reagents to covalently attach the dye to the surface and the
removal of the same afterward as well as a limited control of the
post functionalization efficiency.
EXPERIMENTAL PART
■
Materials. The following reagents were purchased from
commercial suppliers and used without further purification: hydrazine
hydrate (100%, Acros), 4-hydroxybenzaldehyde (≥98%, Merck), 2,4-
dimethylpyrrole (97%, Acros), boron trifluoride diethyl etherate
(minimum 46.5% BF3, Alfa Aesar), triethylamine (≥99.5%, Fluka),
2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 98% Alfa Aesar),
hydrobromic acid (47%, VWR tech grade), phenothiazine (98+%, Alfa
Aesar), chlorosulfonic acid (99%, Aldrich), methyl viologen dichloride
(98%, Sigma-Aldrich), sodium hydrogen carbonate powder (99.5%,
WTL Laborbedarf), sodium iodide (99.5%, Sigma-Aldrich), sodium
sulfate (anhydrous puriss., Sigma-Aldrich), 11-bromo-1-undecanol
(97%, Alfa Aesar), methacryloyl chloride (97%, Alfa Aesar), sodium
carbonate (99.5%, Deutero), phthalimide potassium salt 98% (Merck),
2,6 di-tert-butyl-p-cresol (Fisher Scientific), 2,2′-azobis(2-methylbutyr-
onitrile) (V59, Wako), sodium iodide for analysis (Merck), potassium
carbonate (anhydrous, 99%, Alfa Aesar), trifluoroacetic acid (99.5+%,
Alfa Aesar), and magnesium sulfate extra pure anhydrous (Merck).
Dry solvents (with water content smaller than 30 ppm) were
purchased from Acros. All other solvents were p.a. or HPLC grade and
purchased from different suppliers. Deuterated solvents for NMR
measurements were supplied by Aldrich. Styrene (St) and methyl
methacrylate (MMA) were purchased from Merck as synthesis grade
and were distilled under reduced pressure and stored at 4 °C under
argon.
A versatile tool for the fabrication of functionalized
nanoparticles by (co)polymerization or for the encapsulation
of a great variety of substances is the miniemulsion (co)-
polymerization.30,31 In difference to the conventional emulsion
polymerization, no diffusion step is involved in the polymer-
ization by the addition of an (ultra)hydrophobe to the oil
phase, which results in an osmotic pressure inside the droplets
that is counteracting the Laplace pressure and thus suppressing
the net diffusion. Also, the particle nucleation mechanism is
different: while in emulsion polymerization homogeneous and
micellar nucleation is predominant,32,33 miniemulsion polymer-
ization shows droplet nucleation.30,31,34 Conversely, it means
that the monomer droplet shows compositional and morpho-
logical similarity with the latter polymer particle to some extent
and could thus be seen as a nanoreactor, where the initial
composition of the dispersed phase is preserved in the
particles.30 This is especially of high interest when a
hydrophobic dye should be incorporated or copolymerized;
due to the low water solubility of the fluorescent dye, it is
impossible to use a batch emulsion process where the
monomer needs to diffuse to the polymerization side.25
Nevertheless, all heterophase copolymerizations with func-
tional comonomers have the drawback that not only the surface
of the particles is functionalized, but depending on the
hydrophilicity of the comonomer, it might be also buried
inside the particles35 or form hairy structures on the particle
surface25,36 or even water-soluble homopolymers,35−38 thus
complicating a prediction of the functionalization density.
An alternative to overcome the aforementioned drawbacks is
the use of a polymerizable surfactant. Polymerizable surfactants,
also known as surfmers,39 which is an acronym of surfactant
and monomer, are a type of substances that can make the
employment of a “normal” surfactant superfluous and lead to a
direct and exclusive functionalization of the particle surface
when used in miniemulsion polymerization, as herein the
interphase between the continuous and dispersed phase is
maintained throughout the polymerization. In batch emulsion
polymerization highly reactive surfmers are likely to be
polymerized in the early stages of the polymerization and
become buried inside the growing particles leading to a
destabilization of the dispersion or forming homopolymers
causing bridging flocculation.40,41 Therefore, the miniemulsion
process in combination with surfmers was already discussed in
the 1990s,42,43 but only little was published44 apart from the
past few years, when more papers emerged in the topic.45−49
Methods. Column chromatography was carried out using silica gel
(Kieselgel 60, particle size 0.040−0.063 mm, 230−400 mesh)
purchased from Fluka. For thin-layer chromatography (TLC)
aluminum plates precoated with 0.2 mm silica gel 60 labeled with a
fluorescents indicator were used (Alugram Sil G/UV 0.2 mm silica gel
with fluorescent indicator, Machery-Nagel, Duren, Germany).
̈
1H and 13C NMR spectra were recorded on Bruker Avance
1
spectrometers with 250 or 300 MHz. For H and 13C NMR spectra
tetramethylsilane was used as an external standard.
ESI mass spectra were obtained from a Q-ToF Ultima 3
spectrometer with LockSpray interface from Micromass (Waters).
Samples were injected with a concentration of ∼1 mg/mL in
methanol.
Surface tension was determined by the DuNouy ring methode,
̈
using a DuNouy ring with a diameter of 10 mm and a wire thickness of
̈
0.3 mm, at 20 °C with a DCAT 21 device (Dataphysics, Filderstadt,
Germany), on a sample volume of ∼2 mL. All values presented were
averaged over ten repetitions of push−pull cycles.
HPLC measurements were performed on an Agilent Technologies
1200 Series device, employing a C4 column of Marcherey-Nagel and a
solvent gradient THF/H2O/0.1% TFA from (0/40/60) to (10/100/
0).
The average hydrodynamic diameter DH of the particles and the
paticle size distribution were measured by dynamic light scattering
(DLS) using a Nanophox PCCS (Sympatec GmbH, Clausthal-
Zellerfeld, Germany) at an scattering angle of 90° and a temperature
of 25 °C. Dispersions were diluted to ∼0.1 wt % with distilled water.
The measurement parameters were set to a count rate of 200 kcps,
measuring time was 100 s for each run, and three repetitions were
conducted. The raw data were plotted in Origin software (OriginLab),
and a gauss fit was done.
B
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The ζ-potential measurements were performed in 10−3 mol/L KCl
solutions on a Malvern Nano-Z device at 25 °C; therefore, latexes
were diluted to a solid content of 0.02%.
Scanning electron microscopy (SEM) images were taken with a
Gemini 1530 (Carl Zeiss AG, Oberkochen, Germany). The samples
were prepared by drop casting of 0.01 wt % dispersions on silicon
wafers.
Confocal laser scanning microscopy (CLSM) was performed with
the TCS SP5X (Leica Mikrosysteme Vertrieb GmbH, Wetzlar)
microscope. Steady-state fluorescence spectra were measured with a
Spex FluoroLog 3 spectrofluorometer. Decays of fluorescence were
recorded with the time correlation single photon counting (TCSPC)
technique (FluoTime 200, PicoQuant GmbH). A cuvette (thickness
10 or 1 mm) with a solution/diluted dispersion was excited by a
supercontinuum pulsed laser SC450-2-PP (10 MHz, ∼1 mW/nm,
pulse duration ∼10 ps) (Fianium, Inc.). Output radiation of the laser
was passed through a 4F monochromator50 to select the needed
wavelength. Typically, the radiation with a bandwidth of 10−15 nm
was used. Right angle geometry of detection was chosen for
fluorescence collection. Glan-Thompson polarizers (for excitation
and detection) were arranged under magic angle condition. An
additional long-pass filter Brightline 519/LP (Semrock Inc.) was
placed in front of a Sciencetech Model 9030 monochromator for
better eliminating scattered light. A counting photomultiplier PMA
165 (PicoQuant GmbH) was used as detector. The fluorescence
11-Aminoundecan-1-ol54 (Compound 3). A solution of 2 (10.03 g,
31.6 mmol) in ethanol (185 mL) was cooled to 0 °C, and hydrazine
hydrate (6.2 mL, 127.7 mmol) was added slowly. After stirring for 15
min at 0 °C the reaction was refluxed for 3 h. The work-up was done
according to Fletcher et al.54 Afterward, the reaction was allowed to
cool to room temperature, diluted with 4 M HCl (200 mL), and
filtered, and the filtrate was washed with DCM (100 mL) before being
basified to pH 14 with concentrated NaOH solution. The water phase
was extracted five times with CH2Cl2 (100 mL), the extracts were
combined and dried over Na2SO4, and the solvent was evaporated to
give the desired compound in 92% yield (5.42 g, 28.9 mmol) as a
white solid. 1H NMR (300 MHz, CDCl3): δ = 3.58 (t, J = 6.6 Hz, 2H,
−CH2OH), 2.65 (t, J = 6.8 Hz, 2H, −CH2NH2), 1.72 (s, 3H, −NH2,
−OH), 1.60−1.47 (m, 2H, −CH2−CH2OH), 1.47−1.10 (m, 16H,
−CH2−). 13C NMR (75 MHz, CDCl3): δ = 62.8 (−CH2OH), 42.3
(−CH2NH2), 33.8 (−CH2−CH2NH2), 33.0 (−CH2−CH2OH), 29.7
(−CH2−), 29.6 (−CH2−), 29.6 (−CH2−), 29.5 (−CH2−), 27.0
(−CH2−), 25.9 (−CH2−). LRMS (ESI-MS m/z): calcd for
C11H26NO+ [M + H]+ = 188.19; found 188.22.
11-Bromoundecan-1-ammonium Bromide55,56 (Compound 4). A
suspension of 3 (10.00 g, 53.4 mmol) in aqueous HBr (concentrated,
40 mL) was stirred at 100 °C overnight, resulting in a dark brown
solution. Cooling to room temperature caused the precipitation of the
crude product as a dark gray solid that was recrystallized from acetone.
The product was obtained in 83% yield (14.78 g, 44.6 mmol) as a pale
1
lifetime (τ) for a single-exponential model or average lifetime (τ) for a
gray solide. H NMR (300 MHz, DMSO): δ = 7.80 (bs, 3H, −NH3),
̅
multiexponential model was estimated with the software FluoFit
3.51 (t, J = 6.7 Hz, 2H, −CH2Br), 2.74 (dd, J = 6.1 Hz, 14.5 Hz, 2H,
−CH2−NH3), 1.86−1.67 (m, 2H, −CH2−), 1.64−1.44 (m, 2H,
−CH2−), 1.42−1.13 (m, 14H, −CH2−). 13C NMR (75 MHz,
DMSO): δ = 38.7 (−CH2NH3), 35.2 (−CH2Br), 32.2 (−CH2−
CH2NH3), 28.8 (−CH2−), 28.8 (−CH2−), 28.7 (−CH2−), 28.5
(−CH2−), 28.1 (−CH2−), 27.5 (−CH2−), 26.8 (−CH2−), 25.8
(PicoQuant GmbH) in agreement with eqs 1−3:
n
t
A e−(t−t )/τ dt′
i
′
I(t) =
IRF(t′)
∑
∫
i
−∞
(1)
i=1
(−CH2−). LRMS (ESI-MS m/z): calcd for C11H25BrN+ [M]+
250.12; found 250.14.
=
n
τ =
̅
f τ
∑
i
i
(2)
(3)
N-(11-Hydroxyundecyl)methacrylamide (Compound 5). In a two-
phase system, consisting of DCM (10 mL) as bottom layer and a
solution of Na2CO3 (0.87 g, 8.25 mmol) in water (6 mL), 11-
bromoundecan-1-ammonium bromide (1.00 g, 3.02 mmol) was
dissolved, and the reaction mixture was brought to 0 °C. Subesquently,
methacryloyl chloride (0.38 mL, 3.89 mmol) was slowly added, and
the reaction was stirred for 30 min at 0 °C and afterward for 4 h at
room temperature. Before separating the layers, water (10 mL) and
DCM (10 mL) were added. The aqueous phase was transferred into
alkaline milieu with saturated Na2CO3 solution and extracted twice
with DCM. The organic layers were combined, laced with BHT as
inhibitor, and washed with saturated Na2CO3 solution and water until
the pH was neutral. The organic phase was dried over Na2SO4, and
subsequently the solvent was evaporated. The crude product was
purified by column chromatography on silica gel (petrol ether/diethyl
ether 4:1, followed by pure diethyl ether) to give compound 5 in 81%
(0.78 g, 2.45 mmol) yield, as polymerization inhibitor a small amount
i=1
Aiτi
fi =
n
∑
i=1 Aiτi
It should be noted that in a subsequent part of this document symbol τ
is always attributed to a single-exponential decay, whereas τ to a
̅
multiexponential one.
The quantum yield (QY) of fluorescence was measured with the
fluorescence standard rhodamine 6G in ethanol (QY = 0.9551), in
agreement with the widely used method.52
Synthesis. All synthesis steps were carried out under water- and
air-free conditions, as inert gas argon was used.
2-(11-Hydroxyundecyl)isoindole-1,3-dione (Compound 2). The
compound was synthesized according to Per
́
ez et al.53 with major
modifications in the work-up. 11-Bromo-1-undecanol (25.00 g, 99.5
mmol) and potassium phthalimide (20.9 g, 112.8 mmol) were
dissolved in dimethylformamide (250 mL) and stirred at 80 °C for 16
h. Afterward, the reaction mixture was allowed to cool to room
temperature and was diluted with EtOAc (250 mL). The organic layer
was washed with equivalent volumes of water several times; the
combined aqueous phases were extracted twice with EtOAc (150 mL)
each. The organic layer was washed with brine twice and dried over
magnesium sulfate, the solvent was evaporated under reduced
pressure, and the crude product was recrystallized from methanol.
The desired compound was obtained as a white crystalline powder in
83% yield (26.2 g, 82.5 mmol): Rf = 0.37 (hexane/Et2O 1:3). 1H NMR
(250 MHz, CDCl3): δ = 7.84 (dd, J = 3.0, 5.5 Hz, 2H, HAr), 7.70 (dd, J
= 3.0, 5.5 Hz, 2H, HAr), 3.65 (m, 4H, −CH2O, −CH2N), 1.77−1.47
(m, 5H, −CH2−CH2N, −CH2−CH2O, −OH), 1.45−1.15 (m, 14H,
−CH2−). 13C NMR (63 MHz, CDCl3): δ = 168.6 (−CO), 133.9
(CAr), 132.3 (CAr), 123.3 (CAr), 63.2 (−CH2OH), 38.2 (−CH2N),
32.9 (−CH2−), 29.6 (−CH2−), 29.5 (−CH2−), 29.5 (−CH2−), 29.5
(−CH2−), 29.2 (−CH2−), 28.7 (−CH2−), 26.9 (−CH2−), 25.8
1
of phenothiazine was added. Rf = 0.68 (Et2O). H NMR (300 MHz,
CDCl3): δ = 5.90 (s, 1H, NH), 5.65 (d, J = 0.8 Hz, 1H, CH=), 5.34−
5.22 (m, 1H, CH=), 3.38 (t, J = 6.9 Hz, 2H, −CH2Br), 3.28 (m, 2H,
−CH2NH), 1.94 (m, 3H, −CH3), 1.90−1.74 (m, 2H, −CH2−), 1.61−
1.46 (m, 2H, −CH2−), 1.40 (m, 2H, −CH2−), 1.25 (s, 12H, −CH2−).
13C NMR (75 MHz, CDCl3): δ = 168.52 (−N−CO), 140.3 (Cq=),
119.2 (=CH2), 39.8 (−CH2N), 34.1 (−CH2Br), 32.9 (−CH2−), 29.6
(−CH2−), 29.5 (−CH2−), 29.5 (−CH2−), 29.4 (−CH2−), 29.3
(−CH2−), 28.8 (−CH2−), 28.2 (−CH2−), 27.0 (−CH2−), 18.8
(−CH3). LRMS (FD-MS m/z): calcd for C15H28BrNO = 317.14;
found 317.8 [M]−.
All further reaction steps were carried out under water-free
conditions and under striked exclusion of light by covering the
equipment with aluminum foil.
Compound 8. Bodipy 8 was synthesized according to the procedure
reported by Liu et al.57 To a solution of 4-hydroxybenzaldehyde (1.12
g, 9.13 mmol) and 2,4-dimethylpyrrole (2.00 mL, 19.8 mmol) in THF
(300 mL) a few drops of TFA were added, and the reaction was stirred
overnight. Complete consumption of the aldehyde was verified by
(−CH2−). LRMS (FD+-MS m/z): calcd for C19H27NO3 [M]−
316.2; found 315.0.
=
C
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TLC. Subsequently, DDQ (2.05 g, 9.0 mmol) dissolved in THF (330
mL) was added dropwise within 40 min, and the reaction was stirred at
ambient temperature for 4 h; meanwhile, the color changed from
orange to red to nearly black. After the addition of dry triethylamine
(54 mL, 388.5 mmol) and 45 min of stirring, BF3·Et2O (54 mL, 588
mmol) was added slowly under ice bath cooling. The reaction was
stirred overnight at room temperature, the conversion was checked by
TLC, and the mixture was filtered through a pad of Celite. The filtrate
was washed with brine twice and dried over Na2SO4, and the volatile
substances evaporated under reduced pressure. The crude product was
redissolved in DCM (300 mL) and washed twice with 0.5 M NaHCO3
solution and subsequently several times with water, dried over
Na2SO4, and concentrated to dryness. Compound 8 was received as a
cherry-red solid in 52% yield (1.60 g, 4.7 mmol) after column
chromatography on silica gel, using petrol ether/EtOAc (v/v 2:1) as
the eluent: Rf = 0.5 (PET/EtOAc 2:1). 1H NMR (300 MHz, CDCl3):
δ = 7.12 (d, J = 8.6 Hz, 2H, CHAr), 6.94 (d, J = 8.6 Hz, 2H, CHAr),
5.98 (s, 2H, CHpyrrol), 2.55 (s, 6H, pyrrol−CH3), 1.44 (s, 6H, pyrrol−
CH3). 13C NMR (75 MHz, CDCl3): δ = 156.4 (CAr), 155.4 (CAr),
143.3 (CAr), 141.9 (CAr), 132.0 (CAr), 129.5 (CAr), 127.3 (CAr), 121.3
(CAr), 116.3 (CAr), 14.7 (pyrrol-CH3). LRMS (FD-MS m/z): calcd for
C19H18BF2N2O− = 339.15; found 339.3 [M − H]−.
pyrrol−CH3), 1.62−1.43 (m, 7H, pyrrol−CH3, −CH2−), 1.44−1.20
(m, 14H, −CH2−). 13C NMR: (75 MHz, MeOD): δ = 171.3 (C
ONH), 161.7 (CHeteroAr−SO3), 159.5 (CArO), 153.5, 146.8 (CAr), 145.3
(CAr), 141.5 (CAr), 141.3 (CAr), 141.3 (Cq=), 134.4 (CAr), 130.9 (CAr),
130.5 (CAr), 127.8 (CAr), 123.5 (CAr), 120.1 (CH2=), 116.5 (CAr), 69.2
(−CH2O−), 40.7 (−CH2N−), 30.9 (−CH2−), 30.7 (−CH2−), 30.6
(−CH2−), 30.6 (−CH2−), 30.5 (−CH2−), 30.4 (−CH2−), 30.3
(−CH2−), 28.0 (−CH2−), 27.1 (−CH2−), 18.8 (CH3−C=), 15.1
(pyrrol−CH3), 14.7 (pyrrol−CH3), 14.1 (pyrrol−CH3), 13.3 (pyrrol−
CH3). LRMS (ESI-MS m/z): calcd for C34H45BF2N3NaO5S [M +
2Na+]+ = 702.29; found 702.30.
Double Sulfonation. The double-sulfonated compound could be
obtained in the same manner as the monosulfonated one; only the
concentration of the chlorosulfonic acid dilution and the amount of
NaHCO3 have to be doubled: Rf = 0.5 (EtOAc/MeOH 2:1). 1H NMR
(250 MHz, MeOD): δ = 7.98 (s, 1H, CONH), 7.22 (d, J = 8.8 Hz,
2H, CHAr), 7.12 (d, J = 8.8 Hz, 2H, CHAr), 5.71−5.60 (m, 1H,
=CHtrans), 5.40−5.29 (m, 1H, =CHcis), 4.07 (t, J = 6.3 Hz, 2H, −CH2−
O), 3.27−3.12 (m, 2H, −CH2−NH), 2.78 (s, 6H, pyrrol−CH3), 1.93
(m, 3H, CH3−C=), 1.88−1.76 (m, 2H, CH2−CH2−O), 1.73 (s, 6H,
pyrrol−CH3), 1.64−1.20 (m, 16H, −CH2−). 13C NMR (63 MHz,
MeOD): δ = 171.2 (CONH), 161.8 (CAr), 156.4 (CAr), 147.2 (CAr),
143.5 (CAr), 141.5 (Cq=), 135.7 (CAr), 132.1 (CAr), 130.5 (CAr), 127.5
(CAr), 120.2 (CH2=), 116.7 (CAr), 69.2 (CH2−O), 40.7 (CH2−NH),
30.6 (−CH2−), 30.6 (−CH2−), 30.5 (−CH2−), 30.4 (−CH2−), 30.4
(−CH2−), 30.3 (−CH2−), 28.0 (−CH2−), 27.1 (−CH2−), 18.8
(CH3−C=), 14.4 (pyrrol−CH3), 13.64 (pyrrol−CH3). ESI: LRMS
(ESI-MS m/z): calcd for C34H45BF2N3NaO5S [M + Na+] = 758.25;
found 758.27.
Compound 9. Potassium carbonate (0.22 g, 1.58 mmol) was dried
under vacuum for 15 min under slight warming. Subsequently, DMF
(70 mL) and 8 (0.45 g, 1.32 mmol) were added; through the resulting
solution argon was bubbled for 30 min. Afterward, 5 (0.42 g, 1.32
mmol) was added as well as small amounts of phenothiazine and
sodium iodide. The reaction solution was stirred for 24 h at 100 °C;
after cooling to room temperature, the reaction solution was poured
into brine (200 mL), and the aqueous phase was extracted with EtOAc
several times. The combined organic fractions were washed with brine
twice and dried over Na2SO4. The crude product was purified by
column chromatography on silica gel, using a gradient of petrol ether/
EtOAc 4:1 (v:v) to 2:1. The product was obtained as a gluing dark red
RESULTS AND DISCUSSION
■
Synthesis of Dyes. In the design of the synthetic route to
the surfmer, it was taken into account that Bodipy dyes are
extremely robust to thermal and chemical2 treatment; never-
theless, the purification of Bodipy dyes is rather tedious. For
this reason it was decided to use a convergent synthesis
strategy, where the Bodipy core is preformed and connected to
the hydrophobic tail that contains already the polymerizable
group in the last step, before the formation of the ionic
headgroup of the surfmer.
1
solid, in 44% yield: Rf = 0.26 (PET/EtAc 2:1). H NMR (300 MHz,
CDCl3): δ = 7.16 (d, J = 8.7 Hz, 2H, CHAr), 7.00 (d, J = 8.7 Hz, 2H,
CHAr), 5.98 (s, 2H, CHpyrrol), 5.79 (s, 1H, NH), 5.70−5.63 (m, 1H,
CH2=), 5.35−5.25 (m, 1H, CH=), 4.01 (t, J = 6.6 Hz, 2H, CH2O),
3.40−3.23 (m, 2H, CH2NH), 2.56 (s, 6H, pyrrol−CH3), 1.97 (m, 3H,
CH3), 1.87−1.73 (m, 2H, O−CH2−CH2), 1.38 (m, 22H, pyrrol-CH3,
CH2). 13C NMR (75 MHz, CDCl3): δ = 168.6 (C(=O)−N), 159.9
(CAr), 155.4 (CAr), 143.5 (CAr), 142.2 (CAr), 140.5 (Cq=), 132.1 (CAr),
129.3 (CAr), 127.0 (CAr), 121.3 (CAr), 119.3 (CH2=), 115.3 (CAr), 68.4
(−CH2−O), 39.9 (−CH2N), 29.8 (−CH2−), 29.7 (−CH2−), 29.7
(−CH2−), 29.5 (−CH2−), 29.5 (−CH2−), 27.2 (−CH2−), 26.3
(−CH2−), 18.9 (CH3−CCH2), 14.8 (pyrrol-CH3). FD: LRMS (FD-
MS m/z): calcd for C34H46BF2N3O2 = 577.37; found [M+] = 577.2.
Compounds 10a and 10b. In a small Schlenk tube Bodipy 9 (0.35
g, 0.60 mmol) was dissolved in dry DCM (14 mL), and some crystals
of BHT were added as a radical scavenger. In parallel, chlorosulfonic
acid (0.16 mL, 2.4 mmol) was diluted with DCM (10 mL).
The synthesis of the hydrophobic tail with the polymerizable
group is shown in Scheme 1. The starting point of the synthesis
is the commercially available 11-bromo-1-undecanol (1), which
is converted to 11-amino-1-undecanol54 in a Gabriel synthesis,
employing the Ing−Mansk58 variation. By treatment of
compound 355,56 with boiling hydrobromic acid, the alcohol
group is converted to bromine; additionally, the amine is
Monosulfonation. Subsequently, the reaction flask was immersed
in a ice/salt mixture and cooled to −25 °C; the diluted chlorosulfonic
acid (2.62 mL, 0.63 mmol) was added dropwise over a period of 15
min. The reaction was stirred at −25 °C for 30 min and afterward
allowed to warm to RT, stirring further for 1 h. During that time the
sulfonated product starts to precipitate; by addition of methanol (15
mL) the reaction mixture becomes homogeneous, and the proton was
replaced by a sodium ion. Therefore, NaHCO3 (0.11 g, 1.24 mmol)
was added and the reaction stirred for 20 min.
Scheme 1. Synthesis of the Hydrophobic Tail Containing the
Polymerizable Unit
a
The solvents were removed under reduced pressure, and the
received solid was purified by column chromatography on silica gel. To
elute the product, a gradient of EtOAc/MeOH 16:1 (v:v) to 5:1 (v:v)
was used. The title compound 10a was obtained as a red powder, in
1
50% yield (0.21 g, 0.30 mmol): Rf = 0.38 (EtOAc/MeOH 5:1). H
NMR (300 MHz, MeOD): δ = 7.27−7.15 (m, 2H, CHAr), 7.14−7.05
(m, 2H, CHAr), 6.15 (s, 1H, CHpyrrol), 5.69−5.60 (m, 1H, CH=), 5.34
(m, 1H, CH=), 4.05 (t, J = 6.4 Hz, 2H, −CH2−O), 3.22 (t, J = 7.1 Hz,
2H, −CH2−N), 2.75 (s, 3H, pyrrol−CH3), 2.52 (s, 3H, pyrrol−CH3),
1.93 (m, 3H, CH3−C=), 1.87−1.76 (m, 2H, −CH2−), 1.72 (s, 3H,
a
Reagents and conditions: (a) phthalimide potassium salt, DMF, 80
°C, 16 h; (b) hydrazine monohydrate, EtOH, 15 min, 0 C, 3 h reflux;
(c) HBr (concentrated), 100 °C, overnight; (d) methacryloyl chloride,
H2O, DCM, Na2CO3, 30 min 0 °C, 4 h, RT.
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protonated, so that a quaternization reaction is prevented. The
introduction of the polymerizable group to the hydrophobic tail
is realized under Schotten−Baumann conditions.
The amide linkage between the polymerizable group and the
alkyl spacer seems to be beneficial as methacrylamides are less
likely to hydrolyze or polymerize under various synthetic
conditions.
The Bodipy core 8 with a phenolic moiety in the meso
position was synthesized according to a procedure reported
earlier by Liu et al.57 The acidic phenolic OH group at the
Bodipy core and the nucleophilic substitutable bromine at the
polymerizable alkyl unit allow one to link the Bodipy dye with
the hydrophobic, polymerizable unit in a convergent synthesis
step by Williamson ether formation. The synthesis is shown in
Scheme 2. The Williamson ether formation has proven its
Table 1. Synthesis and Characterization of Polystyrene
Nanoparticles
dye/
amount,
mg
ξ-
potential,
mV
surfactant/
amount, mg
% solid
content
a
sample
DH, nm
b
PS1
SDS/72
9/4.5
−64.3
−58.2
−70.0
−68.0
−54.9
102 17
210 29
251 28
103 17
98 14
20.9
9.5
c
PS2
10a/1.5
10b/1.3
10a/8.8
10b/8.4
c
PS3
9.6
b
PS4
SDS/72
SDS/72
21.4
20.8
b
PS5
a
Hydrodynamic diameter estimated by DLS. Solid content was
determined gravimetrically. Amount of components in miniemulsion
b
polymerization. 24 g of water, 6 g of styrene, 250 mg of hexadecane,
and 100 mg of initiator V59; amounts of SDS and dyes are displaced in
c
the table. 2.4 g of water, 300 mg of styrene, 34 mg of hexadecane, and
14 mg of V59.
Scheme 2. Synthesis Route of Mono- or Double-Sulfonated
Bodipy Surfmer
a
for 10a and γ = 48.0 mN/m for 10b (solutions were prepared
by addition of 1.5 or 1.3 mg of dyes to 2.4 g of water as used for
the miniemulsion polymerization of PS2 and PS3). The
polymerization leads to a stable dispersion with an average
particles diameter of 250 nm (measured by DLS) and rather
high negative ξ-potential (after performed dialysis). As can be
seen from the data in Table 1, the dyes 10a and 10b can
effectively stabilize nanoparticles after the miniemulsion
process. In order to reduce the particle size the surfmers 10a
or 10b were used in combination with sodium dodecyl sulfate
as anionic surfactant. In fact, copolymerization of the dyes in
presence of SDS leads to a considerable reduction of particle
size (down to 100 nm, as it is shown in Table 1). Additionally,
miniemulsion polymerizations were carried out with the
nonsurface active polymerizable dye 9 as a control experiment.
In this experiment dye 9 was dissolved in the organic phase
prior to emulsification.
To proof the copolymerization of surfmers, the dispersions
were freeze-dried; subsequently, the polymer was dissolved in
THF and subjected to HPLC analysis. Only traces of the free
surfmer are detectable in polymeric samples, meaning that most
of the surfmer is incorporated to the polymeric backbone.
Photophysical Properties of Dyes in Solutions. Table 2
summarizes the spectroscopic properties of the synthesized
a
Table 2. Photophysical Characteristics of Synthesized Dyes
Reagents and conditions: (a) TFA, THF, RT, overnight; (b) DDQ,
THF, RT, 4 h; (c) triethylamine, RT, 45 min; (d) BF3·Et2O, 30 min, 0
°C, RT overnight; (e) 5, K2CO3, NaI, phenothiazine, DMF, 100 °C,
24 h.
λmax of abs,
nm/molar
extinction,
L/(mol cm)
λmax of
fluorescence, fluorescence quantum
dye
solvent
nm
lifetime, ns
yield, %
a
9
toluene 503/
8.8 × 104
514
3.2
69
applicability to Bodipy’s containing a phenolic moiety in the
meso position for incorporation into more complex structures
before.59,60
b
10a
water
493/
510
510
1.4
6
4.7 × 104
b
In the final step, the hydrophilic headgroup was created by
either mono- or double-sulfonation of the Bodipy core in the 2-
or 2,6-position with chlorosulfonic acid. The substitution of the
hydrogens in the 2- and 6-position has been already used to
create a couple of water-soluble Bodipy derivatives without any
surface activity.4,61−63
Miniemulsion Copolymerization. Dye-labeled nanopar-
ticles were synthesized via free radical polymerization in
miniemulsion (all ratios of the chemical components are
presented in Table 1) using the hydrophobic initiator V59. The
compounds 10a and 10b show the interfacial activity even at
such low amounts as used for the miniemulsion polymer-
izations. The values of surface tension drop to γ = 48.5 mN/m
10b
water
497/
6.7 × 104
0.29
∼1
a
b
τ. τ.
̅
dyes. Absorption, steady-state fluorescence spectra and decays
of fluorescence in different solvents are presented in Figures 1a
and 1b. Compound 9 is insoluble in water and well soluble only
in organic solvents. Absorption (λmax = 503 nm) and emission
maxima (λmax = 514 nm) are as expected for dyes possessing
the similar Bodipy core. The molar extinction coefficient is
relatively large (8.8 × 104 L/(mol cm)). The decay of
fluorescence recovered from time-resolved measurements
(Figure 1b) was fitted by a single-exponential function and
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Figure 1. (a) Normalized fluorescence (dashed lines) and absorption (solid lines) spectra of dye 9 in toluene (black lines); dye 10a in H2O (red
lines); dye 10b in H2O (blue lines). Fluorescence spectra are recorded at concentration 1 × 10−6 mol/L; absorption at 5 × 10−6 mol/L. (b) Decays
of fluorescence: dye 9 in toluene (black lines), dye 10a in H2O (red lines), dye 10b in H2O (blue lines); the instrument response function of the
setup (IRF) (gray line).
Figure 2. (a) Concentration dependence of the normalized lifetime in case of water solutions of 10a (red circles) and 10b (blue circles) and toluene
solution of 9 (black circles). τ′ is the lifetime measured at a concentration c = 10−6 mol/L: 1.4 ns (10a), 0.29 ns (10b), and 3.2 ns (9). (b)
Concentration dependence of surface tension in case of water solutions of 10a (red circles) and 10b (blue circles). (c) Lifetime of water solution 10b
(c = 1 × 10−5 mol/L) at different amounts of Lutensol AT50 added. Solid red line is a guide to the eye only.
the fluorescence lifetime estimated as τ = 3.2 ns. The QY is
moderately high and is estimated as 69%. In contrast to
compound 9, dyes 10a and 10b are well soluble in water. Dye
10a is additionally partially soluble in nonpolar organic solvents
as toluene and styrene. Absorption spectra in water have
smaller molar extinction coefficients and are hypsochromically
shifted with respect to spectra of dye 9 in toluene. It was found
that an aqueous environment provides significant altering of the
fluorescence properties. The QY of the fluorescence is
dropping down with increasing number of sulfo groups.
Thus, fluorescence of 10a in aqueous solution shows a
expected) on concentration of solutions. In the experiment an
increase of the lifetime is observed for dye 9 in toluene at
concentrations higher than 5 × 10−5 mol/L. This effect can be
ascribed as reabsorption of emitted photons in solutions with
high optical density and subsequent secondary emission that
increases the apparent lifetime of the excited state.64 Thus, the
experiment shows a response of the lifetime on the setup
geometry (mainly the length of the optical pathways for
absorbed and emitted photons) when concentrated solutions
are investigated. Additionally, it was supposed that any other
reason (nether self-quenching or other cooperative phenom-
ena) can be responsible for lifetime changing in toluene
solution of hydrophobic compound 9. In contrast, the solution
of monosulfonated dye 10a in water shows a decreasing lifetime
with increasing dye concentration. We believe that such
behavior reflects the process of micelle formation, and thus,
the cmc (critical micelle concentration) can be very
approximately placed in the diapason of the concentration
range 10−5−10−4 mol/L. In fact, micelle formation leads to
shortening of the interchromophore distance and amplifies the
process of self-quenching. For a more precise estimation of
cmc, we investigated the concentration dependence of the
surface tension. The results shown in Figure 2b indicate that
the value of cmc for surfmer 10a can be estimated as 9 × 10−6
mol/L.
double-exponential decay with an average lifetime of τ = 1.4
̅
ns and the QY of 6%. The double-sulfonated dye in water
demonstrates only weak fluorescence (QY ∼ 1%) and
extremely short lifetime of τ = 0.29 ns.
̅
In general, the modification of the Bodipy core with sulfo
groups does not change the fluorescent properties in the same
way as in our experiments. Fluorescence lifetime and quantum
yield of sulfonated and nonsulfonated derivatives show
unimportant differences.1 Therefore, in order to obtain
additional information about intermolecular interactions in
solutions of dye 9, 10a, and 10b, the concentration dependence
of the fluorescence lifetime was tested. In Figure 2a, the
dependence of normalized lifetime (normalization factor τ′ is
the lifetime measured at a concentration c = 10−6 mol/L when
negligible effect of intermolecular interaction or association are
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Figure 3. (a, c, e) CLSM micrographs of the particles adsorbed onto the surface of microscope slides. (b, d, f) Corresponding SEM micrographs. (a,
b) Dispersion PS1; (c, d) dispersion PS2; (e, f) dispersion PS4. Scale bar is 1 μm for all CLSM images and 250 nm for all SEM images.
Experiments with solutions of compound 10b demonstrate a
very strong influence of solvent polarity on the fluorescent
properties. For instance, the lifetime of 10b measured in less
polar solvents is significantly longer: τ = 2.97 ns in ethanol and
τ = 4.31 ns in a mixture of DMF/chloroform (1/1). It is very
likely that the ultrashort fluorescence lifetime in water reflects
the response of an excited state on very polar media. Solutions
with concentrations down to 1 × 10−7 mol/L were investigated,
and no changes in lifetime were detected. Accordingly to this
fact, the lifetime shortening due to association of the dye
molecule can be excluded. On the other hand, the increase of
the dye concentration gives a stronger raise of normalized
lifetime as compared to dye 9. It should be noted that micelle
formation can make possibly both association of molecules and
changing polarity of media in proximity of dyes. The media
becomes more hydrophobic. The cmc value for surfmer 10b
was estimated from surface tension measurements as 8 × 10−4
mol/L (Figure 2b). Therefore, it can be hypothesized that the
increase of lifetime in the case of 10b also might be attributed
to micelle formation.
found no influence of scattering effects on the fluorescent
lifetime. Considering intermediate values of the lifetime
(ranging between a very short lifetime in water and a rather
long lifetime within the polymeric nanoparticles), the existence
of preferentially surface labeling of nanoparticles can be
assumed when fluorescence surfmers 10a and 10b are used.
The multiexponential behavior of the lifetime might indicate a
distribution of the dye in the outer layer of the polymeric
particles (some molecules settle down closer to the particles
center than others) and the presence of gradient of polymer
hydration within the outer layer. Copolymerization of the dyes
10a and 10b in the presence of SDS leads to a further increase
of the lifetime (up to τ = 3.6 ns for 10a (dispersion PS4) and τ
̅
̅
= 3.9 ns for 10b (dispersion PS5).
There is a direct correlation between the position of the dye
(water phase, water/polymer interface, or polymeric matrix)
and its lifetime:
water
10b
PS3
10b
PS5
10b
τ
̅
< τ
< τ
< τ9PS1
̅
̅
To prove this idea, the following experiment was performed.
Interestingly, the addition of small amounts of the nonionic
surfactant Lutensol AT50 to an aqueous solution of dye 10b
recovers its fluorescent properties (the dependence between
the Lutensol AT50 concentration and the fluorescence lifetime
of 10b is presented in Figure 2b). The fluorescence lifetime was
estimated as τ = 4.02 ns in a solution containing 1 × 10−5 mol/
L of dye 10b and 18 mg/L (7.3 × 10−6 mol/L) of Lutensol
AT50. This result represents a strong hydrophobic interaction
between 10b and the nonionic surfactant and suggests that
micelle formation can lead to a lifetime increase.
An explanation for the different behavior of 10a and 10b in
the micellar solution might be derived from the difference in
the charge density around the Bodipy core, thus influencing the
packing density and preventing stacking in the case of 10b.
Photophysical Properties of the Dyes Copolymerized
via Miniemulsion Process. Miniemulsion copolymerization
of the hydrophobic precursor 9 (dispersion PS1) did not
modify strongly its spectral characteristics; however, the
fluorescence lifetime becomes significantly longer (τ = 5.5
ns). The obtained result indicates the spectral properties of the
dye incorporated covalently within the polymeric nanoparticles.
The fluorescence lifetime of dyes 10a (dispersion PS2) and
The lifetime is importantly longer for molecules located near
the nanoparticle interface than in water but shorter in respect
to the dye distributed inside a solid polymeric matrix. The fact
that the lifetime in case of dispersion PS3 is shorter than in the
case of dispersion PS5 allows one to assume a more
hydrophobic character of the environment in proximity of the
fluorophore molecules when SDS is used as additional
surfactant. Thus, SDS might in some degree shield the contacts
between dyes 10a/10b and water molecules that will be
confirmed additionally by quenching tests.
In whole, extension of fluorescence lifetime in all described
dispersions and very likely increased QY provide bright
fluorescent nanoparticles easily resolved by CLSM (Figure 3).
Figures 3a, 3c, and 3e display the CLSM images of
nanoparticles adsorbed on top of microscope slides. Also,
micrographs in Figures 3b, 3d, and 3f show the particles
morphology as detected by SEM microscopy.
To prove the fact of preferable localization of the surfmers in
the surface layer of the nanopraticles, quenching experiments
were performed with fine dialyzed dispersions. Initially, the
iodine anion (I−) was used which is a well-known quencher due
to the heavy atom effect. Increasing amounts of NaI dissolved
in the continuous phase lowered the lifetime and steady-state
intensity. The fluorescence lifetime is dropping down in the
range from τ = 0.73 ns (without a quencher) to τ = 0.42 ns at
10b (dispersion PS3) are modified in the same way (τPS2 = 1.3
̅
ns and τPS3 = 0.73 ns) and show a double-exponential behavior.
̅
̅
̅
The dispersions were tested with different concentrations of the
dispersed phase (from almost transparent solution at solid
content 0.001% to slightly milky one at solid content 0.1%) and
0.5 M concentration of NaI in case of PS3 and from τ = 3.9 ns
̅
to τ = 2.05 ns in case of PS5. This experiment likely reflects the
process of dynamic quenching, and therefore, experimental
̅
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Figure 4. Quenching of fluorescence in case of dispersions PS1 (red circles), PS3 (blue circles), and PS5 (black triangles). The black lines reflect the
result of fitting according to eq 4. Quenchers: sodium iodide (a) and methyl viologen dichloride (b).
results can be fitted in agreement with the Stern−Volmer
Only a very weak quenching effect was observed for
dispersions PS2 and PS4 even in aqueous solutions of MV2+
with concentrations up to 10−3 mol/L. Since dye 10a is soluble
in water as well as in organic solvents, miniemulsion
copolymerization results in a distribution of the dye between
the surface and volume of the particle. Moreover, the
orientation of the monosufonated Bodipy unit at the particles
interface might differ from double-sulfonated one, as shown in
Scheme 3. Thus, direct interaction of a quencher and the π-
electronic system of the dye 10a might be restricted.
equation:
τ0
τ
= 1 + kqτ0[Q] = 1 + KD[Q]
(4)
The equation provides values of the Stern−Volmer quenching
constant KD = 1.55 L/mol (dispersion PS3) and KD = 1.91 L/
mol (dispersion PS5). Numbers of bimolecular quenching
constant, calculated in agreement with eq 4, were estimated as
kq = 2.06 × 109 L/(mol s) (dispersion PS3) and kq = 4.87 ×
108 L/(mol s) (dispersion PS5). Such relatively low values of
the constants can be explained by two reasons. First, polymeric
nanoparticles have much slower diffusion rates in comparison
to molecules in solutions. Second, the surfaces of the
nanoparticles are negatively charged (Table 1) that leads to
an additional electrostatic repulsion between nanoparticles and
the negatively charged quencher I−. Additionally, a shielding
effect of SDS, as noted earlier, might be responsible for a lower
value of the bimolecular quenching constant in the case of
dispersion PS5.
Scheme 3. Probable Orientation of Dyes 10a and 10b on the
Surface of Nanoparticles after Miniemulsion Polymerization
In further experiments, the quenching effect of methyl
viologen (MV2+) dichloride (Figure 3b) was investigated. Since
the quencher is positively charged, it can interact more
efficiently with the dyes and quench them. Additionally, a
superposition of the dynamic and static quenching in case of
MV2+ provides very high quenching efficiency in micelles,65
solutions,66 and in proximity of an interface.67 In the
CONCLUSIONS
experiments the fluorescence lifetime was changing from τ =
■
̅
0.73 ns (without a quencher) to τ = 0.38 ns at 3.3 × 10−6 mol/
In the present work, new fluorescence surfmers with out-
standing properties were synthesized. The new molecules joint
within their chemical structure different abilities: to be
polymerizable, to emit photons, and to show interfacial activity.
The miniemulsion polymerization performed with participation
of the new surfmers is a direct one-pot procedure for the
production of nanoparticles selectively fluorescence-labeled at
the interface. Without additional synthesis and purification
steps aqueous dispersions of hydrophobic nanoparticles were
obtained showing a strong fluorescent response to substances
dissolved in the continuous phase. In the case of the double-
sulfonated surfmer a strong quenching of particles fluorescence
was observed by sodium iodide or methyl viologen dissolved in
water media. Thus, these nanoparticles can be interesting for
sensing applications based on fluorescence quenching or
fluorescence resonance energy transfer effects. Additionally, a
strong influence of polarity on the fluorescence lifetime was
found for the investigated surfmers. Therefore, processes
̅
L concentration of MV2+ in the case of PS3 and from τ = 3.9 ns
̅
to τ = 2.56 ns at 5 × 10−4 M of MV2+ in the case of PS5. Thus,
̅
abnormally high values of the Stern−Volmer quenching
constant were observed for dispersion PS3 KD = 2.7 × 105
L/mol, which judges about impact of static mechanism
additionally to collision quenching. For dispersion PS5 the
value KD = 688 L/mol was calculated. The important detail was
obtained by investigation of quenching of dispersion PS1. No
traces of interaction were detected between the quencher and
the dye even at high concentration of quencher NaI (Figure 3a)
or methyl viologen dichloride (Figure 3b). Thus, the dye was
effectively protected by the polymeric matrix. This clear
contrast in quenching behavior of dispersions PS1 (from one
side) and PS3 and PS5 (from another side) makes it possible to
conclude that the fluorescent surfmer molecules 10b are
primarily located at the surface if used as comonomers in
miniemulsion polymerization.
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(25) Holzapfel, V.; Musyanovych, A.; Landfester, K.; Lorenz, M. R.;
changing interfacial polarity can be investigated by using the
newly synthesized nanoparticles. For instance, absorption of
synthetic or natural macromolecules onto particles interface or
particles association/aggregation might be quantified. These
issues are currently examined in our laboratory.
Mailander, V. Macromol. Chem. Phys. 2005, 206 (24), 2440−2449.
̈
(26) Allard, E.; Larpent, C. J. Polym. Sci., Part A: Polym. Chem. 2008,
46 (18), 6206−6213.
(27) (a) Borisov, S. M.; Mayr, T.; Mistlberger, G.; Klimant, I. In
Advanced Fluorescence Reporters in Chemistry and Biology II;
Demchenko, A. P., Ed.; Springer: Berlin, 2010; Vol. 9, pp 193−228.
(b) Nikiforow, J.; Konig, A. M.; Langhoff, A.; Pohl, K.; Turshatov, A.;
Johannsmann, D. Langmuir 2010, 26 (16), 13162−13167. (c) Crespy,
D.; Zuber, S.; Turshatov, A.; Landfester, K.; Popa, A.-M. J. Polym. Sci.,
Part A: Polym. Chem. 2012, DOI: 10.1002/pola.25875.
(28) Clark, H. A.; Kopelman, R.; Tjalkens, R.; Philbert, M. A. Anal.
Chem. 1999, 71 (21), 4837−4843.
AUTHOR INFORMATION
Corresponding Author
*Ph +49 6131/379-493; Fax +49 6131/379-330; e-mail
turshat@mpip-mainz.mpg.de.
■
Notes
The authors declare no competing financial interest.
(29) Ji, J.; Rosenzweig, N.; Griffin, C.; Rosenzweig, Z. Anal. Chem.
2000, 72 (15), 3497−3503.
(30) Landfester, K. Macromol. Rapid Commun. 2001, 22 (12), 896−
936.
ACKNOWLEDGMENTS
A.T. and S.B. acknowledge support by the EU-funded FP-7
project EphoCell (No. 227127).
■
(31) Schork, F. J.; Luo, Y.; Smulders, W.; Russum, J. P.; Butte,
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A.;
Fontenot, K. Adv. Polym. Sci. 2005, 175, 129−255.
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Particles; Springer: Berlin, 2005; Vol. 175, pp 1−128.
(33) Gilbert, R. G. Emulsion Polymerization: A Mechanistic Approach;
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