Synthesis of azide-functionalized nanoparticles by microemulsion polymerization and surface modification by click chemistry in aqueous medium

Article abstract of DOI:10.1002/pola.25035

Stable translucent aqueous suspensions of azide-functionalized cross-linked nanoparticles (NPs), with diameters in the 15-20 nm range, were prepared using two synthetic approaches. Copolymerization of azidomethylstyrene (VBN ₃), styrene, and di

Full text of DOI:10.1002/pola.25035

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Synthesis of Azide-Functionalized Nanoparticles by Microemulsion
Polymerization and Surface Modification by Click Chemistry
in Aqueous Medium
Karima Ouadahi, Emmanuel Allard, Birgit Oberleitner, Chantal Larpent
Institut Lavoisier de Versailles UMR-CNRS 8180, Universite´ de Versailles-Saint-Quentin-en-Yvelines,
78035 Versailles Cedex, France
Correspondence to: E. Allard (E-mail: Emmanuel.Allard@uvsq.fr) or C. Larpent (E-mail: Larpent@chimie.uvsq.fr)
Received 10 June 2011; accepted 4 October 2011; published online 22 October 2011
DOI: 10.1002/pola.25035
ABSTRACT: Stable translucent aqueous suspensions of azide-func-
tionalized cross-linked nanoparticles (NPs), with diameters in the
15–20 nm range, were prepared using two synthetic approaches.
Copolymerization of azidomethylstyrene (VBN3), styrene, and
divinylbenzene in various oil-in-water microemulsions led to NPs
with modulable azide contents (0.53–0.78 mmol/g) and surface
over volume distributions. Surface modifications of reactive
NPs bearing chlorobenzyl groups, produced by microemulsion
copolymerization of vinylbenzylchloride, with sodium azide led to
azido-coated NPs with high densities of peripheral groups (0.13–
0.45 mmol/g). It is shown that the nature of the surfactant used
for the preparation of the microemulsion has an impact on the
incorporation of VBN3 in the polymer particles as well as on
the surface reaction yield. The azide-functionalized NPs were
used as clickable polymeric scaffolds for the grafting of sparingly
water-soluble dansyl and fluorescein derivatives through
copper(I)-catalyzed azide-alkyne cycloaddition in water in the
presence of surfactants as solubilizing agents to produce fluores-
C
V
cent NPs.
2011 Wiley Periodicals, Inc. J Polym Sci Part A:
Polym Chem 50: 314–328, 2012
KEYWORDS: functionalization of polymers; copolymerization;
fluorescent nanoparticles; click chemistry; surfactants; azide-
functionalized nanoparticles; microemulsion; aqueous media
INTRODUCTION Nanoparticles (NPs) have attracted consider-
able interest over the past decade due to their special prop-
erties that can be exploited in a wide range of applications
such as in material science,1 catalysis,2 and biomedicine.3
Most of these applications require careful design of the NPs
and reliable strategies for constructing multifunctional
nanomaterials. Indeed, the properties of NPs are not only
governed by their bulk composition but are also greatly
influenced by their surface constitution. For example, in
many practical applications of inorganic NPs, the surface
must be covered with ligands which both prevent aggrega-
tion and also provide a handle that conveniently allows
peripheral functionalization.4 The development of methodol-
ogies that can be used to modify surfaces of NPs in a selec-
tive and efficient way has been pursued. ‘Click Chemistry’
fulfils these criteria. The Click concept was first introduced
in 2001 by Sharpless and co-workers5 with the objective to
establish a set of powerful, highly reliable and selective reac-
tions in synthetic chemistry. The copper catalyzed Huisgen
1,3-dipolar cycloadditions of azides and terminal alkynes,
which were reported independently by Meldal6 and Sharp-
less,7 are archetypical examples of Click Chemistry.
The Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) has
several advantages: (1) it may be performed in water,
buffered media, or mixtures of aqueous/organic solvents and
in the presence of oxygen; (2) usually it is high yielding and
generates no by-products; (3) it is highly regiospecific afford-
ing only the 1,4-disubstituted 1,2,3 triazole isomer; (4) it
tolerates the presence of a wide variety of other functional
groups. As a result, the CuAAC reaction has been used to
modify the surface of a variety of inorganic NPs.8–12
CuAAC has also been applied successfully in polymer
science.13 This reaction can indeed be combined with the
majority of polymerizations processes including controlled
radical polymerization (CRP),14–16 ionic polymerizations,17,18
ring-opening metathesis polymerization,19 polyaddition20 as
well as free CRP.21 Beside the synthesis of polymers by click-
type process,22 the area in which the click concept has demon-
strated the most impact is the functionalization of pre-existing
polymers. The combination of click chemistry and controlled/
living radical polymerization has been found successful to
prepare a variety of polymeric functional nanomaterials with
well-defined structures.23 CRP has opened efficient and
versatile synthetic routes to well-defined amphiphilic block
copolymers, functionalized with alkynyl or azide residues,
which self-assemble in water in a variety of nanostructures
Additional Supporting Information may be found in the online version of this article.
C
V
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like micelles or vesicles.23,24 The CuAAC reaction has been
found a useful tool to cross-link, and hence, to stabilize these
polymeric self-assemblies.23,25 It has also enabled the conjuga-
tion of labeling molecules or biomolecules to polymer vesicles
or micelles.23,26–28
functionalized NPs in the 15–20 nm diameter range. Copoly-
merization with azidomethylstyrene, studied under different
experimental conditions, enables the introduction of the
azide reactive groups both in the core and in the shell of
NPs. The synthesis of azide-functionalized NPs is also readily
achieved by performing surface modifications of previously
prepared cholorobenzyl-NPs with sodium azide. This
approach leads to NPs in which the azide units are located
on the particles surface. Experiments performed in the pres-
ence of different surfactants clearly evidence the prominent
role of the surfactant nature on the functionalization yield.
The azide-functionalized NPs were used as clickable polymer
scaffolds to graft hydrophilic or hydrophobic alkynes by
using CuAAC reaction giving access to fluorescent NPs with
modulable dyes loads. The whole process from the prepara-
tion to the functionalization of azide NPs by CuAAC has been
performed in water without any added organic co-solvent.
Only few examples of CuAAC functionalization of polymer
particles prepared by polymerization in dispersed media
have been reported. Some clickable alkyne and azide-func-
tionalized particles have been obtained by surface modifica-
tion of latexes produced by emulsion polymerization and
used for the grafting of fluorescent probes by CuAAC reac-
tions.29 The preparation of clickable polymer NPs with diam-
eters lower than 50 nm produced by mini- or microemulsion
free radical polymerizations and their post-functionalization
by CuAAC has been scarcely studied. Aylott and Chan30 have
described the preparation of clickable water-dispersible poly-
acrylamide NPs by copolymerization of ethynyl- or azido-ac-
rylamide derivatives in inverse microemulsions which were
then readily modified by CuAAC reaction to afford sensing
nanomaterials. Fre´chet and co-workers31 reported the syn-
thesis of PEGylated NPs by CuAAC on azido-functionalized
polystyrene NPs produced by Oil-in-water (o/w) micro-
emulsion copolymerization with 4-azidomethylstyrene.
This star polymer was used as a platform to elaborate
water-dispersible biomimetic supported catalysts.
Compared to previously reported polyacrylamide-based click-
able nanomaterials, cross-linked polystyrene-based NPs, not
swollen in water, have the relevant advantage of allowing
a discrimination between surface and core in aqueous
medium, and therefore, open the way to well-defined core-
shell type multifunctional nanomaterials. The two synthetic
routes developed in this study lead to structurally different
particles with azide groups located only on the surface
(post-functionalization route) or distributed in the core and
on the surface (copolymerization route). In aqueous medium,
coupling through CuAAC occurs on the surface and leads to
shell-functionalized NPs leaving the particle core available
for further functional arrangements. In further steps, out of
the scope of this article, the particle core can be used as a
host matrix to encapsulate guest molecules by impregna-
tion38 or alternatively, one may use the internal azide groups
as chemical handles to link functionalities within the particle
core by performing CuAAC in an organic solvent that swells
the polystyrene matrix.
In this work, we investigated different strategies to prepare
aqueous suspensions of azido-functionalized polystyrene NPs,
so-called nanolatexes NL, by o/w microemulsion polymeriza-
tion and their use as clickable scaffolds in an all water-based
process without using any organic solvent. o/w microemul-
sion free radical polymerization has proved to be an efficient
and versatile technique to produce ultra-small polymer NPs
from standard commercially available monomers.32–34 A vari-
ety of microemulsions recipes, using nonionic, cationic, or ani-
onic surfactants with or without additional cosurfactants have
been used.32–34 Highly functionalized NPs have been obtained
in one step by copolymerization with a functional comonomer
or in two steps after post-functionalization of primary reactive
NPs.33–41 In microemulsion, copolymerization processes, the
size of the NPs, the amount of functional comonomer incorpo-
rated in the NPs and the core/shell distribution of the
functionalities have been found to depend on the comonomer
partition in between oil droplets/interface/water phase, and
hence on the microemulsion composition, as well as on the
nature of the radical initiator.34–37 The nature of the surfactant
used for the preparation of the starting reactive NPs has also
been found to influence the post-functionalization yield
depending on its interactions with the reactants.34,36 However,
the presence of surfactant has been found beneficial for post-
functionalizations whatever the nature of the reactant and
essential for performing surface modifications with hydropho-
bic water-insoluble reactants.36,40 In the latter case, the
surfactant ensures the micellar solubilization of the reactant
and allows one to perform reactions in aqueous medium with-
out the need of adding organic solvents.
EXPERIMENTAL
Materials
Sodium dodecylsulfate (SDS, Sigma-Aldrich, 98%), dodecyltri-
methylammonium bromide (DTAB, Fluka, 98%), pentan-1-ol
(Acros, 99%), sodium dodecylbenzenesulfonate (DBS, Fluka),
polyethylene glycol mono-4-nonylphenyl ether [NPOEn,
Sigma-Aldrich (n ¼ 12), TCI (n ¼ 5 and 15)], 2,2-dimethoxy-
2-phenyl-acetophenone (DMPA, Acros, 99%), ammonium
persulfate (Acros, 98%), tetramethyldiaminomethane (Fluka,
97%), hydrogen peroxide (Acros, 35 wt % solution in water),
ascorbic acid (Acros, 99%), sodium azide (Acros, 99%),
copper (II) sulfate anhydrous (Fischer, 98%), and sodium
ascorbate (Acros, 99%). Vinylbenzylchloride [VBCl, Aldrich, mix-
ture of isomers meta (70%) and para (30%), 95%], and
divinylbenzene (DVB, Fluka, mixture of isomers meta and para,
80%) were freed of inhibitors by passing through a LC-Si Solid
Phase Extraction tube (Aldrich, cat. no. 57116 and 57133).
Styrene (Acros, 99%) was distilled under reduced pressure.
MilliRo Water and MilliQ water were used for microemulsion
polymerization and sample dilution, respectively.
We show herein that these two strategies are efficient to
obtain stable and transparent aqueous suspensions of azide-
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4-Vinylbenzylazide 1,42 sulfonate derivative 2,43 dansyl deriv-
ative 3,44 and fluorescein derivative 445 were prepared
according to the previously published procedures. Details of
the experimental procedures and characterization are
provided in Supporting Information.
19.6/22.2/4.9 mmol/0.75 mmol, respectively). Magnetic
stirring of the resulting mixture led to a milky emulsion. A
15-wt % aqueous solution of NPOE15 (170 g) was then grad-
ually added until a transparent microemulsion of low viscos-
ity was obtained. The freshly prepared microemulsion was
transferred into a reactor and degassed with nitrogen for 20
min. The polymerization was then performed under white
light irradiation using two 60 W lamps at room temperature
under nitrogen for 12 h. Stable translucent nanolatex was
obtained. Chemical composition: C, 88.05; H, 9.74; N, 2.21%
corresponding to an azide content of 0.53 mmol/g of poly-
mer. Mean diameter (QELS): 20 nm (PDI: 0.066). IR (KBr,
thin film, cmꢁ1): 3,081, 3,060, 3,025, 2,918, 2,866, 2,784,
2,091, 1,696, 1,603, 1,511, 1,495, 1,450, 1,329, 1,230, 1,142,
890, 789, and 699.
Methods
The Infra-Red spectra were recorded using a FTIR apparatus
(Nicolet 550). Elemental analyses, with a precision of 60.3%,
have been obtained from the Service Central d’Analyse (CNRS,
Vernaison, France) and the Service de Microanalyse (ICSN,
Gif-sur-Yvette, France). UV/Vis analyses were performed on a
Perkin–Elmer spectrophotometer UV/Vis/NIR Lambda 19.
Steady-state fluorescence measurements were recorded on a
spectrofluorimeter FluoroMax-3 equipped with a 150 W
Xenon lamp and a slit width of 5 nm. For all measurements
the samples were diluted so that the maximum of absorbance
is less than 0.1 to avoid reabsorption artefact. The mean
hydrodynamic diameter and polydispersity of the NPs were
measured by Quasi-Elastic Light Scattering with a Brookhaven
90Plus. The samples were diluted one hundred times in
MilliRo water then filtered through a 0.22 lm membrane to
remove dust. For each sample, the measurement was per-
formed three times at a temperature of 25 ꢀC with a detection
angle of 90ꢀ.
P3. Experimental details of the synthesis are provided in Sup-
porting Information. Chemical composition: C, 87.65; H, 9.13;
N, 3.21% corresponding to an azide content of 0.76 mmol/g of
polymer. Mean diameter (QELS): 17 nm (PDI: 0.077).
Synthesis of co-NPN3_(NPOEn) in Nonionic Surfactants-Based
Microemulsion Using the Redox Couple H2O2/Ascorbic
Acid as Radical Initiator P4
An aqueous solution of sodium DBS (2-wt %, 1.43 g) was
added to a mixture of monomers (styrene/DVB/vinylbenzy-
lazide 1: 17.5/17.3/6 mmol, respectively) and NPOE5
(0.72 g). Magnetic stirring of the resulting mixture led to a
milky emulsion. A 15-wt % aqueous solution of a mixture of
NPOE12/NPOE20 (50/50 wt %, 157 g) was then gradually
added until a transparent microemulsion of low viscosity
was obtained. The resulting microemulsion was transferred
Separation of the Resulting Polymers for IR and
Elemental Analyses
The polymers were isolated and purified using the following
procedure: the polymer was flocculated by adding 20 mL of
methanol to 2 g of suspension and separated by centrifuga-
tion (8000 rpm, 15 min). The resulting white precipitate was
extensively washed with demineralized water (stirring for
into
a three-necked round bottom flask and degassed
ꢀ
1 h followed by centrifugation) and finally dried at 50 C.
with nitrogen for 20 min. Hydrogen peroxide (35 wt %
in water, 24.7 mmol) and ascorbic acid (aqueous solution
0.615 mol/L, 1.23 mmol) were added successively to the
microemulsion. The reaction mixture was then heated at
35 ꢀC under nitrogen for 30 min. Stable translucent nanola-
tex was obtained. Chemical composition: C, 87.46; H, 9.62; N,
2.90% corresponding to an azide content of 0.69 mmol/g of
polymer. Mean diameter (QELS): 15 nm (PDI: 0.053). IR
(KBr, thin film, cmꢁ1): 3,083, 3,054, 3,025, 2,921, 2,860,
2,091, 1,603, 1,507, 1,494, 1,453, 1,349, 1,294, 1,244, 1,115,
952, 827, and 700.
Synthesis of Azide-Functionalized NPs by
Copolymerization (co-NPN3
)
Synthesis of co-NPN3_(DTAB) in DTAB-Based Microemulsion
Using DMPA as Radical Initiator P1
A microemulsion was prepared in a reactor by progressive addi-
tion, upon gentle magnetic stirring, of a mixture of monomers
(styrene/DVB/vinylbenzylazide 1:20.2/20.8/5 mmol, respec-
tively) and 2,2-dimethoxy-2-phenyl acetophenone (DMPA, 0.74
mmol) to 132 g of a 15 wt % solution of DTAB in demineralized
water. The resulting microemulsion was degassed with nitrogen
for 20 min and the polymerization was then performed under
white light irradiation using two 60 W lamps at room tempera-
ture under nitrogen for 24 h. Stable translucent nanolatex was
obtained. Polymer particle content in suspension is 3.7 wt %.
Chemical composition: C, 88.22; H, 7.61; N, 3.26% corresponding
to an azide content of 0.78 mmol/g of polymer. Mean diameter
(QELS): 14 nm (PDI: 0.027). IR (KBr, thin film, cmꢁ1): 3,084,
3,059, 3,026, 2,924, 2,852, 2,097, 1,602, 1,510, 1,493, 1,452, 904,
797, 760, and 700.
Synthesis of Azide-Functionalized NPs by Post-
Fonctionalization (Post-NPN3
)
The synthesis of primary reactive chlorobenzyl-functional-
ized NPs NPCl was obtained following the previously
described procedures.36,38,39 Details of the experimental
procedures are provided in Supporting Information.
P5
Chemical composition: C, 84.89; H, 7.26; Cl, 3.10% corre-
sponding to a chloride content of 0.87 mmol/g of polymer.
Mean diameter (QELS): 15 nm (PDI: 0.036).
Synthesis of co-NPN3_(NPOEn) in Nonionic Surfactants-Based
Microemulsion Using DMPA as Radical Initiator P2
An aqueous solution of sodium DBS (2-wt %, 1.67 g) was
added to NPOE5 (0.83 g) and a mixture of monomers con-
taining the initiator (styrene/DVB/vinylbenzylazide 1/DMPA:
P5-bis. Chemical composition: C, 86.24; H, 7.37; Cl, 1.55%
corresponding to a chloride content of 0.44 mmol/g of poly-
mer. Mean diameter (QELS): 17 nm (PDI: 0.05).
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P6. Chemical composition: C, 78.38; H, 8.21; Cl, 3.10%
corresponding to a chloride content of 0.87 mmol/g of poly-
mer. Mean diameter (QELS): 17 nm (PDI: 0.089).
3,083, 3,058, 3,025, 2,924, 2,853, 1,602, 1,510, 1,493, 1,452,
1,235, 1,157, 990, 905, 837, 798, 761, and 701.
Synthesis of Dansyl-Functionalized NPs
P7. Chemical composition: C, 83.77; H, 7.36; Cl, 3.69% cor-
responding to a chloride content of 1.04 mmol/g of polymer.
Mean diameter (QELS): 35 nm (PDI: 0.086).
A typical procedure is given for one set of NPs. All the others
were prepared following this procedure, details are provided
in Supporting Information.
P13
Synthesis of Azide-Functionalized NPs from
Dansyl derivative 3 (0.0423 g, 0.147 mmol) was added to
15 g of an aqueous suspension of post-NPN3 (P8). The mix-
ture was stirred at room temperature until complete solubili-
zation of 3 (30 min). Then, 1 mL of a freshly prepared
aqueous solution of CuSO4 (0.0189 g, 0.118 mmol) and
sodium ascorbate (0.263 g, 1.33 mmol) were added. The
resulting suspension was stirred at room temperature for
4 days. The suspension was dialyzed against aqueous solu-
tion of DTAB (15 wt % then 5 wt %) using an ultrafiltration
device (Vivascience, Vivaspin concentrator 20, 50,000 MWCO
PES). The process was repeated until no dansyl derivative
was detected in the filtrate. Chemical composition: C, 84.31;
H, 7.11; N, 2.22; S, 0.59% corresponding to a dansyl content
of 0.18 mmol/g of polymer. Mean diameter (QELS): 18 nm
(PDI: 0.125). IR (KBr, thin film, cmꢁ1): 3,086, 3,059, 3,025,
2,926, 2,846, 2,788, 2,098, 1,941, 1,867, 1,802, 1,682, 1,633,
1,602, 1,509, 1,491, 1,451, 1,331, 1,269, 1,232, 1,167, 1,143,
1,032, 992, 903, and 845.
Chlorobenzyl-NPs Prepared in DTAB-Based
Microemulsion Post-NPN3_(DTAB) P8
Azide-coated NPs NPN3 were prepared by adding an aqueous
solution of sodium azide (3.39 g in 10 mL of water) to
100 g of the freshly prepared crude suspension of NPCl (P5)
the pH of which was previously adjusted to 7.5. The result-
ing suspension was stirred at room temperature for 1 week.
The excess of sodium azide was then removed by dialysis
through a porous cellulose membrane (MWCO 12,000–
14,000 D) toward an aqueous solution of DTAB (15 wt %)
leading to a stable translucent aqueous suspension of azide-
functionalized NPs (NPN3). Polymer particle content in
suspension is 4.6 wt %. Chemical composition: C, 84.77; H,
7.77; N, 1.91% corresponding to an azide content of
0.45 mmol/g of polymer. Mean diameter (QELS): 16 nm
(PDI: 0.037). IR (KBr, thin film, cmꢁ1): 3,080, 3,055, 3,022,
2,917, 2,852, 2,094, 1,937, 1,867, 1,800, 1,698, 1,600, 1,509,
1,495, 1,448, 1,346, 1,263, 1,070, 1,030, 987, 907, and 834.
P8-bis. Chemical composition: C, 84.83; H, 7.48; N, 2.11%
corresponding to an azide content of 0.50 mmol/g of polymer.
Mean diameter (QELS): 17 nm (PDI: 0.053).
P14
From P8; chemical composition: C, 83.44; H, 7.11; N, 2.51; S,
1.02% corresponding to a dansyl content of 0.32 mmol/g of
polymer. Mean diameter (QELS): 17 nm (PDI: 0.101).
P8-ter. Chemical composition: C, 82.96; H, 7.17; N, 2.18% cor-
responding to an azide content of 0.53 mmol/g of polymer.
Mean diameter (QELS): 15 nm (PDI: 0.033).
P15
From P8; chemical composition: C, 84.11; H, 7.18; N, 2.67; S,
1.06% corresponding to a dansyl content of 0.33 mmol/g of
polymer. Mean diameter (QELS): 19 nm (PDI: 0.064).
Azide-functionalized NPs NPN3 from NPCl prepared in nonionic
and anionic surfactants-based microemulsions were obtained
using the same procedure as P8. Details of the experimental
procedures are provided in Supporting Information.
P15-bis
From P8-ter; chemical composition: C, 82.99; H, 7.09; N,
2.18; S, 0.92%. corresponding to a dansyl content of 0.29
mmol/g of polymer. Mean diameter (QELS): 19 nm (PDI:
0.084).
Azide-Functionalized NPs Obtained in the Presence of
Non-Ionic Surfactant Post-NPN3_(NPOEn) P9
From P6; dialysis were performed toward an aqueous solu-
tion of NPOE12 (1 wt %). Stable translucent nanolatex was
obtained. Chemical composition: C, 77.68; H, 8.37; N, 0.54%
corresponding to an azide content of 0.13 mmol/g of poly-
mer. Mean diameter (QELS): 17 nm (PDI: 0.037). IR (KBr,
thin film, cmꢁ1): 3,469, 3,081, 3,058, 3,023, 2,919, 2,866,
2,096, 1,723, 1,700, 1,604, 1,515, 1,488, 1,446, 1,346, 1,296,
1,246, 1,112, 946, 904, 831, 792, 758, and 704.
P18
From P9; dialysis were performed toward aqueous solution
of NPOE15 (15 wt % then 5 wt %). Chemical composition: C,
83.08; H, 7.41; N, 1.18; S, 0.29% corresponding to a dansyl
content of 0.09 mmol/g of polymer. Mean diameter (QELS):
16 nm (PDI: 0.074).
P19
P9-bis. Chemical composition: C, 76.78; H, 8.31; N, 0.52%
corresponding to an azide content of 0.12 mmol/g of polymer.
Mean diameter (QELS): 17 nm (PDI: 0.033).
From P9; chemical composition: C, 83.32; H, 7.44; N, 1.21; S,
0.49% corresponding to a dansyl content of 0.16 mmol/g of
polymer. Mean diameter (QELS): 15 nm (PDI: 0.083).
Azide-Functionalized NPs Obtained in the Presence
of Anionic Surfactant Post-NPN3_(SDS) P10
From P7; dialysis were performed toward an aqueous solu-
tion of SDS (1 wt %). Chemical composition: C, 84.19; H,
7.49; N, 0.25% (under precision limit). Mean diameter
(QELS): 34 nm (PDI: 0.083). IR (KBr, thin film, cmꢁ1): 3,446,
P21
From P1; dialysis were performed toward aqueous solution
of DTAB (15 wt % then 1.5 wt %). Chemical composition: C,
81.32; H, 6.92; N, 3.78; S, 1.06% corresponding to a dansyl
content of 0.33 mmol/g of polymer. Mean diameter (QELS):
20 nm (PDI: 0.128).
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P24
80.98; H, 6.97; N, 4.61; S, 0.41% corresponding to fluorescein
content of 0.13 mmol/g polymer. Mean diameter (QELS): 23
nm (PDI: 0.185).
From P3, dialysis were performed toward aqueous solution
of NPOE15 (15 wt % then 5 wt %) Chemical composition: C,
81.13; H, 7.05; N, 4.94; S, 1.59% corresponding to a dansyl
content of 0.50 mmol/g of polymer. Mean diameter (QELS):
17 nm (PDI: 0.066).
P12-bis
Prepared using the procedure described earlier for P12 with
the addition of N,N0-dimethylethylenediamine (6 mol. per
mol CuSO4). Chemical composition: C, 84.68; H, 7.33; N,
1.92; S, 0.22%. Mean diameter (QELS): 16 nm.
P25
From P4; chemical composition: C, 78.59; H, 7.39; N, 4.43; S,
1.65% corresponding to a dansyl content of 0.52 mmol/g of
polymer. Mean diameter (QELS): 16 nm (PDI: 0.05).
Synthesis of Sulfonate-Functionalized NPs
P22
Synthesis of Fluorescein-Functionalized NPs
A typical procedure is given for one set of NPs. All the others
were prepared following this procedure, details are provided
in Supporting Information.
Sulfonate derivative 2 (0.082 g, 0.574 mmol) was added to
10 g of aqueous suspension of co-NPN3 (P1). Then, 1 mL of
a freshly prepared aqueous solution of CuSO4 (0.020 g,
0.116 mmol) and sodium ascorbate (0.260 g, 1.305 mmol)
were added. The resulting suspension was stirred at room
P11
temperature for
4 days. The suspension was dialyzed
Fluorescein derivative 4 (0.066 g, 0.147 mmol) was added to
15 g of an aqueous suspension of post-NPN3 (P8). The pH
was adjusted with NaOH (1 M) until complete solubilization
of 4 (pH close to 7). Then, 1 mL of a freshly prepared aque-
ous solution of CuSO4 (0.0095 g, 0.06 mmol) and sodium
ascorbate (0.131 g, 0.66 mmol) were added. The resulting
suspension was stirred at room temperature for 7 days. The
suspension was dialyzed against aqueous solution of DTAB
(15 wt % then 5 wt %) using an ultrafiltration device (Vivas-
cience, Vivaspin concentrator 20, 50,000 MWCO PES). The
process was repeated until no fluorescein derivative was
detected in the filtrate. Chemical composition: C, 84.58; H,
7.19; N, 1.88; S, 0.14% (under precision limit). Mean diame-
ter (QELS): 16 nm (PDI: 0.087). IR (KBr, thin film, cmꢁ1):
3,084, 3,062, 3,027, 2,915, 1,851, 2,099, 1,944, 1,869, 1,805,
1,700, 1,633, 1,601, 1,513, 1,494, 1,454, 1,347, 1,239, 1,156,
1,114, 1,071, 989, 901, and 835.
through a porous cellulose membrane (MWCO 12,000–
14,000D) toward an aqueous solution of DTAB (15 wt %
then 5 wt %) and led to a stable translucent aqueous sus-
pension of sulfonate-functionalized NPs. Chemical composi-
tion: C, 83.88; H, 7.80; N, 3.12; S, 0.62% corresponding to
sulfonate content of 0.19 mmol/g polymer. Mean diameter
(QELS): 16 nm (PDI: 0.213). IR (KBr, thin film, cmꢁ1): 3,432,
3,089, 3,056, 3,023, 2,921, 2,852, 2,096, 1,687, 1,601, 1,511,
1,491, 1,446, 1,343, 1,230, 1,197, 1,119, 1,033, 988, 903,
837, 792, 764, and 698.
Synthesis of Dual Fluorescent NPs
P26
Fluorescein derivative 4 (0.240 g, 0.54 mmol) was added to
an aqueous suspension of post-NPN3_(DTAB) (P8-bis, 20 g,
polymer content 4.5 wt %). The pH was adjusted with NaOH
(1M) until complete solubilization of 4 (pH close to 7). Then,
1 mL of a freshly prepared aqueous solution of CuSO4 (0.068
g, 0.43 mmol) and sodium ascorbate (0.954 g, 4.8 mmol)
were added. The resulting suspension was stirred at room
temperature for 5 days. Dansyl derivative 3 (0.156 g,
0.54 mmol) was then added to the suspension. The mixture
was stirred at room temperature until complete solubiliza-
tion of 3 (1 h). A freshly prepared aqueous solution (1 mL)
of CuSO4 (0.068 g) and sodium ascorbate (0.954 g) were
added. The reaction mixture was stirred at room tempera-
ture for 6 days. The suspension was dialyzed against
aqueous solution of DTAB (15 wt% then 5 wt%) using an
ultrafiltration device (Vivascience, Vivaspin concentrator 20,
50,000 MWCO PES). The process was repeated until no fluo-
rescein and dansyl derivatives were detected in the filtrate.
Polymer elemental analysis: C, 77.85; H, 6.44; N, 2.48; S,
1.25% corresponding to an overall fluorophores content of
0.39 mmol/g of polymer. Mean diameter (QELS): 18 nm
(PDI: 0.061). IR (KBr, thin film, cmꢁ1): 3,080, 3,061, 3,024,
2,922, 2,850, 2,096, 1,599, 1,493, 1,452, 1,331, 1,233, 1,158,
1,075, 902, and 863.
P12
From P8; chemical composition: C, 81.26; H, 7.24; N, 1.86; S,
0.22% (under precision limit). Mean diameter (QELS): 16 nm
(PDI: 0.091).
P16
From P9-bis; dialysis were performed toward aqueous solu-
tion of NP15 (15 wt % then 5 wt %). Chemical composition:
C, 83.61; H, 7.24 N, 1.11; S, 0.21% (under precision limit).
Mean diameter (QELS): 17 nm (PDI: 0.121).
P17
From P9; polymer elemental analysis: C, 81.76; H, 7.32; N,
1.03; S, 0.16% (under precision limit). Mean diameter
(QELS): 16 nm (PDI: 0.091).
P20
From P1; dialysis were performed toward aqueous solution
of DTAB (15 wt % then 1.5 wt %). Chemical composition: C,
86.16; H, 7.56; N, 3.13; S, 0.32% corresponding to fluorescein
content of 0.10 mmol/g polymer. Mean diameter (QELS):
18 nm (PDI: 0.283).
P27
P23
Synthesis was realized following the general procedure
described earlier, from an aqueous suspension of post_-
NPN3_(DTAB) (P8-bis, 30 g), 4 (0.060 g, 0.134 mmol), CuSO4
From P3; dialysis were performed toward aqueous solution
of NP15 (15 wt % then 5 wt %). Chemical composition: C,
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SCHEME 1 Synthesis of azide-functionalized NPs by copolymerization.
(0.043 g, 0.27 mmol), and sodium ascorbate (0.60 g,
3.03 mmol), then 3 (0.154 g, 0.54 mmol), CuSO4 (0.043 g), and
sodium ascorbate (0.60 g). Polymer elemental analysis: C,
81.68; H, 7.19; N, 2.49; S, 1.01% corresponding to an overall
fluorophores content of 0.32 mmol/g of polymer. Mean diame-
ter (QELS): 19 nm (PDI: 0.1). IR (KBr, thin film, cmꢁ1): 3,082,
3,056, 3,024, 2,919, 2,850, 2,785, 2,094, 1,685, 1,598, 1,511,
1,493, 1,453, 1,323, 1,233, 1,160, 903, 795, 759, and 697.
either with a mixture of nonionic surfactants (nonylphenolpo-
lyethoxylates, NPOEn) or with a cationic surfactant (DTAB). The
o/w microemulsions of mixture of monomers are readily
obtained using simple and versatile methods. The first method
consists of forming a water-in-oil (mixture of monomers) emul-
sion using a low hydrophilic lipophilic balance (HLB) nonionic
surfactant (NPOE5) and then titrating with an aqueous solution
of a high HLB surfactant (NPOE15 or a mixture of NPOE12/
NPOE20: 50/50 wt %) until a clear transparent microemulsion
is obtained.36,46 In the second method, the microemulsion is
obtained upon progressive addition of the mixture of mono-
mers to an aqueous solution of DTAB (15 wt %).37,39–41
Study of Reaction-Time for CuAAC on NPs
P15-bis
The functionalization of azide-functionalized NPs (post-NPN3
)
with dansyl derivative 3 has been performed using the pro-
cedure described earlier for P15. Aliquots of 4 g of the sus-
pension were taken after 12, 24, and 48 h, and the isolated
polymers were characterized by elemental analysis and IR
spectroscopy. Nitrogen and sulfur contents in the polymer:
N, 2.20; S, 0.92% (after 12 h), N, 2.33; S, 0.98% (after 24 h),
and N, 2.18; S, 0.92% (after 48 h). Experimental details and
IR spectra are given in Supporting Information.
The polymerizations were performed under mild conditions
below 35 ꢀC with a water-soluble or an oil-soluble radical ini-
tiator: hydrogen peroxide/ascorbic acid redox system35–36,46
or 2,2-dimethoxy-2-phenylacetophenone (DMPA) decomposed
under white light irradiation.37,39–41 Whatever the experimen-
tal conditions, polymerization of all monomers occurs with
100% conversion within 30 min to a few hours (controlled by
TLC analysis) depending on the initiating system. In every
case, stable translucent aqueous suspensions of nanolatexes
are obtained and remain stable since neither flocculation nor
aggregation of the particles have been observed after more
than 1 year storage. The characteristics of the resulting
polymer particles are given in Table 1. The average particles
diameters, determined by Quasi-Elastic Light Scattering
(QELS), range from 14 to 20 nm with narrow size distribu-
tions. The spectroscopic characterization of the polymers, iso-
lated by flocculation, demonstrates that copolymerization of
the vinylbenzyl azide with styrene and DVB has taken place.
RESULTS AND DISCUSSION
Preparation and Characterization of
Azide-Functionalized NPs
Preparation by Copolymerization in Microemulsion
Azide-functionalized NPs (co-NPN3) were obtained by copoly-
merization of vinylbenzyl azide 1 (VBN3, 10–15% mol) with
styrene (St) and DVB as cross-linking agent (Scheme 1). The
polymerizable azide derivative 1 was prepared by nucleophilic
substitution of vinylbenzyl chloride with sodium azide in
dimethylsulfoxide at room temperature.42 The copolymeriza-
tions have been performed in o/w microemulsions stabilized
The IR spectra show the vibration band of azide groups (vN3
)
at 2,100 cmꢁ1 and the vibration bands of aromatic part
TABLE 1 Nanoparticles co-NPN3 Obtained by Microemulsion Copolymerization with Vinylbenzylazide 1
VBN3
Monomers
Load (wt %)a
Azide Contente
VBN3
Incorporated (%)f
No.
(mol %)
Surfactantb
Initc
D (nm)d
(mequiv./g Polymer)
P1
P2
P3
P4
a
11
11
15
15
4.0
3.2
3.5
3.1
DTAB
I1
I1
I1
I2
14
20
17
15
0.78
0.53
0.76
0.69
87
60
60
58
NPOE15
NPOE12–20
NPOE12–20
e
Monomers load (wt %) in the starting microemulsion.
Deduced from elemental analysis (nitrogen content) with an uncer-
b
c
See text for abbrevations.
tainty of 60.07 mequiv./g.
Molar ratio azide content in polymer/vinylbenzylazide introduced in
the starting mixture of monomers.
f
Radical initiator; I1: DMPA/UV, 20 ꢀC, 24 h; I2: H2O2/ascorbic acid,
30–35 ꢀC, 30 min.
d
Particle diameter determined from QELS.
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SCHEME 2 Synthesis of azide-functionalized NPs by post-functionalization.
(See Fig. S1 in Supporting Information). Elemental analyses
confirm that vinylbenzyl azide has been incorporated. The
azide content in the polymer range from 0.53 to 0.78 mmol/g
and depends on the experimental conditions.
styrene and DVB, according to our previously described
procedures.36,38–41
To evaluate the influence of the nature of the surfactant on
the post-functionalization reaction, copolymerizations have
been performed in microemulsions prepared with nonionic
surfactants (NPOEn),36 with a cationic surfactant (DTAB)38,39
as well as with an anionic surfactant (SDS) in the presence
of pentanol as cosurfactant.35,36 In all cases, stable translu-
cent aqueous suspensions of chlorobenzylated NPs were
obtained. The experimental conditions and main characteris-
tics of the resulting particles are summarized in Table 2. In
microemulsion polymerizations, the size of the resulting par-
ticles depends on the monomer load and on the monomer-
to-surfactant weight ratio.32–34 Accordingly, chlorobenzylated
particles P7 (monomers load: 6.2%, monomers/surfactant:
0.6) are larger than particles P5 and P6 (monomers load
4.3–4.9%, monomers/surfactant: 0.3–0.35). Post-functionali-
zation reactions have been performed under mild conditions
at room temperature by adding an excess of sodium azide
(10 equiv. per chlorine in the polymer) to the freshly as-pre-
pared aqueous suspensions of NPCl without pre-purification.
The suspensions remained stable and translucent during
reaction except when the anionic surfactant was used.
Unreacted sodium azide is easily removed from the suspen-
sion by dialysis at the end of reaction. The particle sizes and
the azide contents deduced from elemental analysis of the
isolated polymer are given in Table 2. The azide contents in
the polymer particles show that the functionalization yield
strongly depends on the surfactant. The amount of grafted
azide groups is negligible in the presence of the anionic sur-
factant while reactions performed in the presence of cationic
or nonionic surfactants lead to azide-functionalized NPs con-
taining 0.13–0.45 mmol N3/g of polymer. Moreover, in the
later cases DLS analyses show that the NP sizes remain
The percentage of VBN3 incorporated in the polymer
particles depends on the nature of the surfactant used to
prepare the microemulsion. For DMPA-initiated copolymer-
ization in cationic surfactant-based microemulsions, more
than 85% of VBN3 initially introduced has been incorporated
(Table 1, P1). Conversely, for copolymerizations performed in
nonionic surfactants-based microemulsions, the incorpora-
tion yield is only about 60% whatever the VBN3 load (10 or
15 mol %) and the initiating system (Table 1, P2–P4). These
results seem to indicate that in nonionic surfactants-based
microemulsions vinylbenzylazide is partitioned between
the aqueous phase and the oil droplets. The loss of VBN3
can be explained if one considers that polymerization of
1 partly takes place in water giving rise to water-soluble low
molecular weight polymers which are removed during the
purification but could not be detected due to their low
concentration. It is worth noticing that the azide content in
the NPs can be modulated either by changing the microemul-
sion type (for a given initial VBN3 load and a given radical
initiator, P1–P2 Table 1) or by changing the VBN3 load (for
a given microemulsion type and a given radical initiator,
P2–P3 Table 1).
Preparation by Post-Functionalization of
Chlorobenzyl NPs
The preparation of azide-coated NPs was then studied using
the two steps procedure by nucleophilic substitution on the
surface of chlorobenzyl-functionalized NPs with sodium
azide (Scheme 2). Reactive benzylhalide-functionalized NPs
were prepared by copolymerizing VBCl (15% mol) with
TABLE 2 Nanoparticles post-NPN3 Obtained by Surface Reactions on Nanoparticles Bearing Chloromethyl Groups
No.
Starting Suspension NPCl
NPN3
Azide
Chlorine
Contente
Contente
wt %a
Surfactantb
Initc
D (nm)d
(meq/g Polymer)
No.
D (nm)d
(mequiv./g Polymer)
P5
P6
P7
a
4.3
4.9
6.2
DTAB
NPOE
SDS
I1
I2
I3
15
17
35
0.87
0.87
1.04
d
P8
16
17
33
0.45
0.13
0.06
P9
P10
Monomers wt % in the starting microemulsion; VBCl content 15 (mol %).
Particle diameter determined from QELS.
b
c
e
See text for abbrevations.
Radical initiator; I1: DMPA/UV, 20 ꢀC, 24 h; I2: H2O2/ascorbic acid,
Determined from elemental analysis with an uncertainty of 60.07
mequiv./g.
30–35 ꢀC, 30 min; I3: (NH4)2S2O8/(CH3)2NACH2AN(CH3)2, 20 ꢀC, 24 h.
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reactions, as it will be demonstrated in the next section. This
simple procedure affords a straightforward access to azide-
functionalized NPs of 16–17 nm mean diameter. The proper
choice of the surfactant used in the first polymerization step
allows one to produce NPs with modulable N3 contents rang-
ing from 0.13 to 0.45 mmol/g of polymer with nonionic and
cationic surfactants, respectively. Anionic surfactants should
be prohibited because they inhibit the surface reaction.
Preparation and Characterization of Functionalized-NPs
by Cu-Catalyzed Azide/Alkyne Cycloaddition (CuAAC)
Reaction
The azide-functionalized NPs, prepared either by post-func-
tionalization (post-NPN3
) or copolymerization (co-NPN3),
FIGURE 1 Size distribution deduced from QELS analysis of
chlorobenzyl-functionalized NPs NPCl (P5, right side), azide-
functionalized NPs NPN3 (P8, middle side) and dansyl-function-
alized NPs NPdansyl (P14, left side).
were then assessed as clickable scaffolds by copper catalyzed
reaction with alkynes 2, 3, and 4 (Scheme 3). The water-
soluble alkyne 2 was prepared in quantitative yield by
nucleophilic substitution of propargyl bromide with sodium
sulfite.43 The hydrophobic fluorescent dansyl derivative 3
was synthesized in 76% yield by reacting dansyl chloride
with propargylamine hydrochloride in the presence of
triethylamine in CH2Cl2.44 The fluorescein derivative 4, a flu-
orescent pH-sensitive dye, was obtained in 80% yield by
unchanged after surface modification (Fig. 1). The grafting of
azide groups is further evidenced by the appearance of the
characteristic N3 stretching band at 2,100 cmꢁ1 in the IR
spectra (Figs. 2a and S2 in the Supporting Information). The
ratios of the area of the N3 and CH stretching bands at 2100
and 2800–3100 cmꢁ1 in polymer particles P8 and P9 are in
good agreement with the highest azide load in P8 obtained
from NPCl in the presence of cationic surfactant (See Fig. S3
in Supporting Information).
The influence of the surfactant on the surface reaction can
be understood considering the interactions between the
reactant Nꢁ3 and the surfactant molecules adsorbed on
the particle surface as well as competitive side reactions. In
the presence of an anionic surfactant, electrostatic repulsions
with the negatively charged surfactant molecules adsorbed
on the particles surface take the anionic reagent away from
the surface, thus, accounting for the very poor functionaliza-
tion yield. Conversely, when the reaction is performed in the
presence of a cationic surfactant, electrostatic attractions
with the adsorbed positively charged amphiphilic molecules
favor the approach of the azide anions. The resulting high
local concentration of azide ions associated with the surfac-
tant ammonium units in the interfacial region is expected to
favor the nucleophilic substitution of benzylhalide on the
surface of NPs and accounts for the observed high function-
alization yield. Moreover, in the presence of DTAB, we also
have to consider the exchange of chloride for bromine, which
has been reported to occur during and after the polymeriza-
tion.39 This substitution is beneficial, because it transforms
benzylchloride groups into more reactive benzylbromide
residues. In the presence of nonionic surfactants, the local
concentration of reactive azide anions near the surface is
lower so that hydrolysis of the chlorobenzyl groups may
become competitive, thus, accounting for the lower function-
alization yield. Moreover, in aqueous medium the polysty-
rene-based NPs are not swelled, so that substitution by azide
nucleophile occurs exclusively with the benzylhalide residues
located near the surface which are accessible for surface
FIGURE 2 Infra-red spectra of azide-functionalized NPs (a: P8),
fluorescein-functionalized NPs (b: P12) and dansyl-functional-
ized NPs (c: P15).
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SCHEME 3 Coupling of functional alkynes via CuAAC to azide-functionalized NPs.
reacting fluorescein isothiocyanate with propargylamine
hydrochloride in the presence of triethylamine in dimethylfor-
mamide (DMF).45 All the CuAAC reactions were performed in
water, without any added co-solvent, directly on the aqueous
suspensions of azide-functionalized NPs in the presence of the
surfactant used in the polymerization step (cationic surfactant
DTAB or nonionic surfactant NPOE15) to ensure the micellar
solubilization in the case of poorly water-soluble alkynes 3
and 4. Click reactions were performed in the presence of
Cu(I) catalyst generated in situ from copper sulfate and so-
dium ascorbate (about 0.4 and 4.5 equiv. in relation to azide
content in the polymer, respectively). The reactions were
allowed to proceed for 4–7 days at ambient temperature,
according to reaction times previously used for other post-
functionalization of nanolatexes.38–41 The content of Cu(I) cat-
alyst and times of reaction were not optimized (vide infra). Af-
ter the reaction, the copper catalyst and the unreacted alkyne-
functionalized derivatives were easily removed by exhaustive
dialysis. For fluorescent alkynes, dialyses were performed
until no dyes were detected in the filtrate.
with dyes 3 and 4 to prepare fluorescent NPs. CuAAC
reactions were first performed between the water insoluble
dansyl derivative 3 and azide-coated NPs prepared by post-
functionalization either in the presence of cationic or non-
ionic surfactants. The results reported in Table 3 (See also
Table S3 in Supporting Information) show that the grafting
of the dansyl derivative 3 readily proceeds in water with
good yields, whatever the surfactant. Maximum functionaliza-
tion yields (>80% as deduced from elemental analysis of the
isolated polymers) are achieved in the presence of a stoichio-
metric amount or slight excess of 3 (1 or 2 equiv. relative to
azide content) leading to NPs with dansyl contents ranging
from 0.16 to 0.33 mol/g of polymer, depending on the sur-
factant used in the preparation process (Table 3, entries P14,
P15, and P19). The coupling of the dansyl derivative is
clearly evidenced by the decrease of the azide vibration
band at 2,100 cmꢁ1 in IR spectra of the isolated polymers
(Figs. 2c and S4–S6 in Supporting Information). As detailed
in Supporting Information (Tables S1–S3, Figs. S4–S6), the
surface reaction yield can be estimated, with acceptable
accordance with elemental analyses, from the ratio of the
areas of azide vibration band and CH stretching bands. The
IR spectrum of P15 in Figure 2c shows the almost complete
disappearance of the azide vibration band indicating a close
to quantitative functionalization (estimated yield of 95%) in
the presence of 2 equiv. of alkyne 3. A functionalization yield
of about 80–85% is achieved under stoichiometric conditions
(Table 3 entry P14 and Fig. S5 in Supporting Information).
Noteworthy, reaction performed with 0.5 equiv. of 3 in the
presence of DTAB shows that an efficient grafting, with 80%
The clicking ability of azide-functionalized NPs was first
investigated by reacting the water-soluble alkyne 2 as a
small molecule model. The decrease of the azide vibration
band in the IR spectrum as well as the sulfur content in the
polymer resulting from the reaction with azide-functional-
ized NPs P1, prepared by copolymerization in the presence
of DTAB, demonstrate the grafting of the sulfonate and indi-
cate that the experimental conditions used for the CuAAC
reactions are properly chosen (Table 4, entry P22). The click-
ing ability of azide-functionalized NPs was then assessed
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TABLE 3 Surface Reactions on Nanoparticle Post-NPN3 Via CuAAC
Suspension
(Surfactant)a
Reactant [C]
Bound Reactante
No.
P11
(mM)b/[No. equiv.]c
D (nm)d
(mequiv./g Polymer)
Yield (%)f
10
P8 (DTAB)
4
16
0.04g
0.07g
0.07g
0.18
9.8/[ꢂ0.5]
4
P12
P12-bish
P13
P14
P15
P16
P17
P18
P19
a
P8 (DTAB)
16
16
18
17
19
17
16
17
15
17
19.6/[ꢂ1]
4
(20)
17
P8-ter (DTAB)
P8 (DTAB)
19.6/[ꢂ1]
3
(16)
45
9.8/[ꢂ0.5]
3
P8 (DTAB)
0.32
83
19.6/[ꢂ1]
3
(83)
80
P8 (DTAB)
0.33
39.2/[ꢂ2]
4
(95)
30
P9-bis (NPOE12
)
0.07g
0.05g
0.09
8.7/[ꢂ2.0]
4
P9 (NPOE12
P9 (NPOE12
P9 (NPOE12
)
)
)
24
5.8/[ꢂ1.5]
3
41
3.7/[ꢂ1]
3
(40)
82
0.16
7.4/[ꢂ2]
(80)
Starting suspension.
order of 20%. In brackets: functionalization yield calculated from IR
spectrum.
b
c
Concentration of alkyne in the starting suspension.
No. equiv.: mole alkyne/equiv. azide in the polymer.
Particle diameter determined from QELS.
g
The yield should be handled with care since the elemental analysis
d
e
shows a very low sulfur content, and thus, an estimation of the yield
based on elemental analysis is difficult.
Reaction performed in the presence of N,N0-dimethylethylenediamine
Amount of bound reactant (60.07 mequiv./g) in the final polymer
calculated from the total amount of sulfur in the polymer (elemental
analysis).
h
with a ligand/CuSO4 ratio of 6:1.
f
Functionalization yield calculated from the ratio of nitrogen and
sulfur in the polymer (elemental analysis) with an uncertainty in the
yield relative to the alkyne derivative, is achieved under sub-
stoichiometric conditions (Table 3, entry P13). The evolution
of the functionalization yield as a function of the reaction
time has been studied under the conditions of experiment
P15 (2 equiv. 3, DTAB): elemental analyses and IR spectra of
the polymer isolated after 12, 24, and 48 h presented in
Figure S6 in Supporting Information demonstrate that the
coupling reaction is completed after one night with a close
to quantitative functionalization (experiment P15bis, Table
S3 in Supporting Information).
The grafting of dansyl derivative 3 by CuAAC also readily pro-
ceeds, under similar experimental conditions (using 2 equiv.
of 3 relative to the overall azide content), on azide-functional-
ized NPs prepared by copolymerizations (Table 4). In these
particles, the azide groups are distributed between the core
and the surface, the click reaction occurs only on accessible N3
residues located near the surface, since the polystyrene-based
NPs are not swollen in water. In all cases, the IR spectra of the
isolated polymers show a decrease of the intensity of the N3
vibration band at 2100 cmꢁ1 (Table S2 and Figs. S7–S8 in Sup-
porting Information). The content of grafted dansyl residues,
deduced from elemental analyses, range from 0.33 to 0.52
mmol/g of polymer, depending on the starting suspension
(samples P21, P24, and P25, Table 4).
These results demonstrate that the density of dansyl on the
particle surface can be easily modulated by the proper
choice of the starting azide-functionalized NPs, as well as by
the amount of 3 introduced in the starting suspension. More-
over, as illustrated in Figure 1, QELS analyses of the starting
chlorobenzylated NPs, azide-functionalized NPs and click-
functionalized NPs show that the size distribution is not
affected by the two consecutive functionalization steps
(Fig. 1). The suspensions of the resulting NPs are stable in
the presence of surfactant without any detectable aggrega-
tion after more than 1-year storage.
According to the almost quantitative yields previously
observed with azido-coated particles and considering that in
the present reactions the alkyne reactant 3 has been intro-
duced in excess regarding the accessible N3 groups, one can
assume an almost quantitative functionalization of the
surface azide groups. This assumption allows us to estimate
the amount of accessible azide groups in NPs obtained by
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TABLE 4 Surface Reactions on Nanoparticle co-NPN3 Via CuAAC
Suspension
(Surfactant)a
Reactant [C] (mM)b/
[No. equiv.c]
Bound Reactante
No.
P20
D (nm)d
(mequiv./g polymer)
Yield (%)f
15
P1 (DTAB)
4
18
0.10g
58/[ꢂ2]
P21
P22
P23
P24
P25
a
P1 (DTAB)
3
20
16
23
17
16
0.33
0.19
0.13
0.5
49
26
13
59
73
57.4/[ꢂ2]
2
P1 (DTAB)
57.4/[ꢂ2]
4
P3 (NPOE12–20
P3 (NPOE12–20
P4 (NPOE12–20
)
)
)
24/[ꢂ1]
3
47.9/[ꢂ2]
3
0.52
37/[ꢂ2]
f
Starting suspension.
Functionalization yield calculated from the ratio of nitrogen and sulfur
b
c
Concentration of alkyne in the starting suspension.
No. equiv.: mole alkyne/equiv. azide in the polymer.
Particle diameter determined from QELS.
Amount of bound reactant (60.07 mequiv./g) in the final polymer calcu-
lated from the total amount of sulfur in the polymer (elemental analysis).
in the polymer (elemental analysis) with an uncertainty in the order of
20%.
d
e
g
The yield should be handled with care since the elemental analysis
shows a very low sulfur content and thus an estimation of the yield
based on elemental analysis is difficult.
copolymerizations. Owing to the similar size of the starting
azide-functionalized particles (P1, P3, and P4), the higher
functionalization yields, 60–75% relative to the overall
amount of azide, obtained with NPs prepared in nonionic
surfactants-based microemulsions (Table 4, entries P24, P25)
than with NPs prepared in the cationic surfactant-based
microemulsions (about 50%, Table 4 entry P21) are indica-
tive of higher surface concentrations of reactive azide groups
on NPs prepared in nonionic surfactants-based microemul-
sions than on NPs produced in the cationic surfactant based
microemulsions. An interfacial partition of VBN3 in nonionic
surfactants-based microemulsions (vide supra) may account
for this observed high concentration of azide groups on the
NPs periphery. Noteworthy, the percentage of accessible
azide groups is also dependent on the radical initiating sys-
tem used for the copolymerization process: 60 and 75% for
NPs prepared using the oil-soluble initiator DMPA and the
water-soluble redox initiating system (hydrogen peroxide-
ascorbic acid), respectively.
entry P12-bis). For all the resulting aqueous suspensions of
NPs, the linkage of fluorescein units was confirmed by the
absorption and fluorescence spectra, recorded at neutral pH,
which display the expected features of fluorescein dianionic
form (Figs. 4 and S11–S13 in Supporting Information). In the
presence of nonionic surfactant, the fluorescein absorption
and emission are observed around 495 and 517 nm, respec-
tively (Table 5). In the presence of cationic surfactant, the
absorption and emission bands are slightly shifted, as we
have already reported,41 and are observed around 505 and
525 nm, respectively (Table 5). However, the low sulfur
content in the polymer and the only slight change of the
intensity of the azide vibration band in the IR spectra
(Fig. 2b) indicate that the amount of grafted fluorescein
units are limited. The functionalization yields do not exceed
The absorption and fluorescence emission spectra of all
the prepared translucent aqueous suspensions dansyl-
functionalized NPs display the characteristic spectroscopic
features of dansyl units with a maximum absorption wave-
length around 335–340 nm and a maximum fluorescence
emission around 495–510 nm depending on the nature of
the surfactant (Fig. 3, Table 5 and Figs. S9–S10 in Support-
ing Information).
As illustrated by the results reported in Tables 3 and 4, the
grafting of the fluorescein derivative 4 by CuAAC is less
efficient, whatever the amount of alkyne derivative and the
starting azide-functionalized NPs. The use of a ligand (N,N0-
dimethylethylenediamine), known to improve the catalytic
efficiency,47 has no effect on the coupling yield (Table 3,
FIGURE 3 Absorption (a) and fluorescence emission (b) spectra
of aqueous suspension of NP_dansyl (P13) at neutral pH in the
presence of 0.5 wt % of DTAB. Dansyl dye was excited at 340 nm.
324
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TABLE 5 Maximum Absorption and Emission Wavelengths of
Aqueous Suspensions of Dansyl-Functionalized Nanoparticles
and Fluorescein-Functionalized Nanoparticles
Absorption
Max. (nm)
Emission
No.
Dye
Surfactant
Max. (nm)
P13
P21
P24
P11
P20
P16
P23
a
Dansyl
DTAB
338
338
335
503
505
497
495
509 (340)a
501 (340)a
496 (340)a
525 (500)a
527 (505)a
518 (497)a
516 (495)a
Dansyl
DTAB
Dansyl
NPOE15
DTAB
Fluorescein
Fluorescein
Fluorescein
Fluorescein
DTAB
NPOE15
NPOE15
FIGURE 4 Absorption (a) and fluorescence emission (b) spectra
of aqueous suspension of NP_fluorescein (P11) at neutral pH in
the presence of 0.5 wt % of DTAB. Fluorescein dye was excited
at 500 nm.
Excitation wavelength (nm).
10–15% with maximum fluorescein loads in the particles
lower than 0.15 mmol/g of polymer (Tables 3 and 4).
To determine whether the difference of reactivity observed
with the dansyl and the fluorescein derivatives 3 and 4
arises from surface effects, we studied model reactions with
benzylazide as a molecular model mimic of the surface azide
groups (See Scheme S1 in Supporting Information). The reac-
tion of 3 with benzylazide readily proceeds in water/THF
medium in the presence of CuSO4 and sodium ascorbate and
leads to the expected adduct in 70% yield. On the contrary,
under the same experimental conditions, the reaction with 4
does not give significant isolable amounts of cycloadduct.
Changing the solvent, the amounts of copper sulfate and
sodium ascorbate, using a Cu(I) salt and also incorporating
ligand such as tris-(benzyltriazolylmethyl)amine (TBTA)47
led to similar results (See Table S4 in Supporting Informa-
tion). The limited amount of fluorescein derivative that can
be grafted by click reaction on azide-functionalized NPs
therefore appears to be a consequence of the poor reactivity
of the alkyne reagent 3 rather than the result of unappropri-
ate experimental grafting conditions. Although limited, the
quantity of grafted fluorescein units is high enough to pro-
duce fluorescent nanolatexes.
Fluorescent dual NPs are readily prepared by two consecu-
tive CuAAC reactions with 4 then 3 without intermediate
separation. Given that the coupling of fluorescein is much
less efficient than the coupling of dansyl, dual NPs with ad-
justable dansyl to fluorescein ratio cannot be achieved in a
single step by mixing the alkyne derivatives. However, tak-
ing profit of the close to quantitative grafting of the dansyl
derivative 3, one can control the dansyl to fluorescein ratio
by coupling first fluorescein and in a second step dansyl in
adjustable amount. Owing to the poor efficiency of the cou-
pling of fluorescein, this approach is nevertheless limited to
dual NPs with low fluorescein contents. The syntheses have
been performed on azide-coated NPs (P8-bis) prepared by
post-functionalization in the presence of DTAB (Scheme 4).
Two experiments have been performed with different
amounts fluorescein and dansyl derivatives 4 and 3: a reac-
tion with a slight excess of both dyes (P26: 1.2 equiv. 4
and 1.2 equiv. 3 per azide unit) and a reaction with sub-
stiochiometric amounts of both dyes (P27: 0.8 equiv. 4 and
0.2 equiv. 3).
SCHEME 4 Synthesis of dual NPs.
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sodium azide with reactive chlorobenzylated NPs, produced
by microemulsion copolymerization, affords an easy access
to azide-coated NPs from standard monomers. The proper
choice of the surfactant allows one to control the density of
azide groups grafted onto the surface. The highest function-
alization yields are achieved in the presence of a cationic
surfactant. The clickable azide-functionalized NPs readily
undergo copper catalyzed cycloadditions with alkynes in
aqueous suspensions giving access to fluorescent NPs with
modulable amounts of grafted dyes. The click reaction has
been found particularly efficient (with almost quantitative
yields) for the grafting of a sparingly water-soluble dansyl
derivative, the presence of surfactant allowing its micellar
solubilization.
The two synthetic routes to azide-functionalized NPs (post-
functionalization and copolymerization) lead to structurally
different particles regarding the distribution of the clickable
units, and hence, each of them presents specific interests and
offers different perspectives for the elaboration of functional
nanomaterials. The post-functionalization route leads to NPs
where all the azide groups are located on the surface and
accessible for coupling in aqueous medium. These azide-
coated NPs are particularly well suited to prepare shell-
functionalized NPs, the coupling being easily evidenced by IR
spectroscopy. These particles might be valuable templates for
the elaboration of fluorescent sensing devices by embedment
of fluorophores within the particle core.41,48 The copolymer-
ization route leads to NPs in which the azide groups are
located both in the core and on the surface. One benefit of
this approach is the possibility to modulate both the content
and the core/surface distribution of azide groups. In aqueous
medium, coupling through CuAAC occurs on the surface and
leads to shell-functionalized NPs leaving the internal azide
groups available for further chemical modifications. Azide-
functionalized NPs prepared by copolymerization might
therefore open the way to dually functionalized core-shell
type nanomaterials by performing a second functionalization
step in organic medium that will involve the azide groups
located within particle core. Owing to the wide scope of
applicability of click chemistry, the all-water-based synthetic
approaches depicted here should therefore give straightfor-
ward access to a variety of functional polymer NPs.
FIGURE 5 Absorption spectra of aqueous suspension of dual
NPs (P26: solid lines and P27: dashed lines). Measurements
were performed at pH ¼ 7.5 in the presence of 0.5 wt % of DTAB.
Spectroscopic studies of the resulting nanolatexes and analy-
ses of the resulting isolated polymers demonstrate that both
dyes have been grafted on the NPs in both experiments. The
IR spectrum of the isolated polymer particles prepared with
slight excesses of 4 and 3 (P26) shows the almost complete
disappearance of the azide vibration band (See Fig. S14 in
Supporting Information) which is an indicative of an almost
quantitative reaction. Elemental analysis confirms this result
with an estimated reaction yield of about 80–90% with an
overall dye load of about 0.4 mmol/g of polymer. As
expected, the reaction performed in the presence of substio-
chiometric amounts of reactants (P27) leads to lower dyes
load (about 0.3 mmol/g). The absorption spectra of the
aqueous suspensions of dual-functionalized NPs recorded at
neutral pH in the presence of 0.5 wt % DTAB display the
absorption bands of the dansyl and fluorescein dyes at 331
and 503 nm, respectively (Fig. 5). The ratio of dansyl to fluo-
rescein grafted on the NPs was estimated from the measured
values of absorbance to be around 80/20 and 95/5 for P26
and P27, respectively. The fluorescence spectra show the
emission of both dyes around 500–525 nm. A detailed spec-
troscopic study of the fluorescence properties of these dual
NPs will be published elsewhere.
CONCLUSION
This study demonstrates that stable aqueous suspensions of
azide-functionalized polymer NPs in the 15–20 nm diameter
range are readily obtained either in one step by micro-
emulsion copolymerization with an azido-functionalized
comonomer or in two steps by post-functionalization of
chlorobenzylated NPs. Microemulsion copolymerization with
azidomethylstyrene leads to NPs with azide groups distrib-
uted between the particle core and the surface. The overall
azide content and the percentage of accessible azide groups
depend on the microemulsion composition, mainly the
nature of the surfactant used, and on the radical initiator.
The highest concentration of peripherical accessible azide
groups is obtained when polymerizations are conducted in
nonionic surfactants-based microemulsions. The reaction of
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